Influenza A 2009 pandemic H1N1 polypeptide fragments comprising endonuclease activity and their use

ABSTRACT

The present invention relates to polypeptide fragments comprising an amino-terminal fragment of the PA subunit of a viral RNA-dependent RNA polymerase possessing endonuclease activity, wherein the PA subunit is from Influenza A 2009 pandemic H1N1 virus or is a variant thereof. This invention also relates to (i) crystals of the polypeptide fragments which are suitable for structure determination of the polypeptide fragments using X-ray crystallography and (ii) computational methods using the structural coordinates of the polypeptide to screen for and design compounds that modulate, preferably inhibit the endonucleolytically active site within the polypeptide fragment. In addition, this invention relates to methods of identifying compounds that bind to the PA polypeptide fragments possessing endonuclease activity and preferably inhibit the endonucleolytic activity, as well as the compounds themselves. Preferably, the compounds are identifiable by the methods disclosed herein or the pharmaceutical compositions are producible by the methods disclosed herein.

CROSS REFERENCES

This application is a divisional application to U.S. patent applicationSer. No. 13/635,064, filed on Sep. 14, 2012, which is a National Stagefiling of International Application Serial No. PCT/EP2011/001274, filedMar. 15, 2011, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/340,335 filed Mar. 16, 2010, the disclosures of which areexpressly incorporated herein by reference.

TECHNICAL FIELD OF INVENTION

The present invention relates to polypeptide fragments comprising anamino-terminal fragment of the PA subunit of a viral RNA-dependent RNApolymerase possessing endonuclease activity, wherein said PA subunit isfrom Influenza A 2009 pandemic H1N1 virus or is a variant thereof. Thisinvention also relates to (i) crystals of the polypeptide fragmentswhich are suitable for structure determination of said polypeptidefragments using X-ray crystallography and (ii) computational methodsusing the structural coordinates of said polypeptide to screen for anddesign compounds that modulate, preferably inhibit theendonucleolytically active site within the polypeptide fragment. Inaddition, this invention relates to methods identifying compounds thatbind to the PA polypeptide fragments possessing endonuclease activityand preferably inhibit said endonucleolytic activity, preferably in ahigh throughput setting. This invention also relates to compounds whichare able to modulate, preferably to inhibit, the endonuclease activityof the PA subunit polypeptide fragment or variant thereof of the presentinvention and pharmaceutical compositions comprising said compounds forthe treatment of disease conditions caused by viral infections withviruses of the Orthomyxoviridae family, Bunyaviridae family and/orArenviridae family, preferably caused by viral infections with InfluenzaA 2009 pandemic H1N1 virus. Preferably, said compounds are identifiableby the methods disclosed herein or said pharmaceutical compositions areproducible by the methods disclosed herein.

BACKGROUND OF THE INVENTION

Influenza is responsible for much morbidity and mortality in the worldand is considered by many as belonging to the most significant viralthreats to humans. Annual Influenza epidemics swipe the globe andoccasional new virulent strains cause pandemics of great destructivepower. At present the primary means of controlling Influenza virusepidemics is vaccination. However, mutant Influenza viruses are rapidlygenerated which escape the effects of vaccination. In the light of thefact that it takes approximately 6 months to generate a new Influenzavaccine, alternative therapeutic means, i.e., antiviral medication, arerequired especially as the first line of defense against a rapidlyspreading pandemic.

An excellent starting point for the development of antiviral medicationis structural data of essential viral proteins. Thus, the crystalstructure determination of the Influenza virus surface antigenneuraminidase (von Itzstein et al., 1993, Nature 363:418-423) leddirectly to the development of neuraminidase inhibitors with anti-viralactivity preventing the release of virus from the cells, however, notthe virus production. These and their derivatives have subsequentlydeveloped into the anti-Influenza drugs, zanamivir (Glaxo) andoseltamivir (Roche), which are currently being stockpiled by manycountries as a first line of defense against an eventual pandemic.However, these medicaments provide only a reduction in the duration ofthe clinical disease. Alternatively, other anti-Influenza compounds suchas amantadine and rimantadine target an ion channel protein, i.e., theM2 protein, in the viral membrane interfering with the uncoating of thevirus inside the cell. However, they have not been extensively used dueto their side effects and the rapid development of resistant virusmutants (Magden et al., 2005, Appl. Microbiol. Biotechnol. 66:612-621).In addition, more unspecific viral drugs, such as ribavirin, have beenshown to work for treatment of Influenza infections (Eriksson et al.,1977, Antimicrob. Agents Chemother. 11:946-951). However, ribavirin isonly approved in a few countries, probably due to severe side effects(Furuta et al., 2005, Antimicrob. Agents Chemother. 49:981-986).Clearly, new antiviral compounds are needed, preferably directed againstdifferent targets.

Influenza virus A, B, C and Isavirus as well as Thogotovirus belong tothe family of Orthomyxoviridae which, as well as the family of theBunyaviridae, including the Hantavirus, Nairovirus, Orthobunyavirus,Phlebovirus, and Tospovirus, are negative stranded RNA viruses. Theirgenome is segmented and comes in ribonucleoprotein particles thatinclude the RNA dependent RNA polymerase which carries out (i) theinitial copying of the single-stranded virion RNA (vRNA) into viralmRNAs and (ii) the vRNA replication. For the generation of viral mRNAthe polymerase makes use of the so called “cap-snatching” mechanism(Plotch et al., 1981, Cell 23:847-858; Kukkonen et al., 2005, Arch.Virol. 150:533-556; Leahy et al., 1997, J. Virol. 71:8347-8351; Noah andKrug, 2005, Adv. Virus Res. 65:121-145). The polymerase is composed ofthree subunits: PB1 (polymerase basic protein), PB2, and PA. For thecap-snatching mechanism, the viral polymerase binds via its PB2 subunitto the 5′ RNA cap of cellular mRNA molecules which are cleaved atnucleotide 10 to 13 by the endonucleolytic activity of the polymerase.The capped RNA fragments serve as primers for the synthesis of viralmRNAs by the nucleotidyl-transferase center in the PB1 subunit (Li etal., 2001, EMBO J. 20:2078-2086). Finally, the viral mRNAs are 3′-endpoly-adenylated by stuttering of the polymerase at an oligo-U motif atthe 5′-end of the template. Recent studies have precisely defined thestructural domain of PB2 responsible for cap-binding (Fechter et al.,2003, J. Biol. Chem. 278:20381-20388; Guilligay et al., 2008 Nat.Struct. Mol. Biol. 15:500-506). The endonucleolytic activity of thepolymerase has hitherto been thought to reside in the PB1 subunit (Li etal, supra).

The polymerase complex seems to be an appropriate antiviral drug targetsince it is essential for synthesis of viral mRNA and viral replicationand contains several functional active sites likely to be significantlydifferent from those found in host cell proteins (Magden et al., supra).Thus, for example, there have been attempts to interfere with theassembly of polymerase subunits by a 25-amino-acid peptide resemblingthe PA-binding domain within PB1 (Ghanem et al., 2007, J. Virol.81:7801-7804). Moreover, there have been attempts to interfere withviral transcription by nucleoside analogs, such as2′-deoxy-2′-fluoroguanosine (Tisdale et al., 1995, Antimicrob. AgentsChemother. 39:2454-2458) and it has been shown that T-705, a substitutedpyrazine compound may function as a specific inhibitor of Influenzavirus RNA polymerase (Furuta et al., supra). Furthermore, theendonuclease activity of the polymerase has been targeted and a seriesof 4-substituted 2,4-dioxobutanoic acid compounds has been identified asselective inhibitors of this activity in Influenza viruses (Tomassini etal., 1994, Antimicrob. Agents Chemother. 38:2827-2837). In addition,flutimide, a substituted 2,6-diketopiperazine, identified in extracts ofDelitschia confertaspora, a fungal species, has been shown to inhibitthe endonuclease of Influenza virus (Tomassini et al., 1996, Antimicrob.Agents Chemother. 40:1189-1193). However, the inhibitory action ofcompounds on the endonucleolytic activity of the viral polymerase washitherto only studied in the context of the entire trimeric complex ofthe polymerase.

The PA subunit of the polymerase is functionally the leastwell-characterised, although it has been implicated in both cap-bindingand endonuclease activity, vRNA replication, and a controversialprotease activity. PA (716 residues in influenza A) is separable bytrypsination at residue 213. The recently determined crystal structureof the C-terminal two-thirds of PA bound to a PB1 N-terminal peptideprovided the first structural insight into both a large part of the PAsubunit, whose function, however, still remains unclear, and the exactnature of one of the critical inter-subunit interactions (He et al.,2008, Nature 454:1123-1126; Obayashi et al., 2008, Nature454:1127-1131). Systematic mutation of conserved residues in the PAamino-terminal domain have identified residues important for proteinstability, promoter binding, cap-binding and endonuclease activity ofthe polymerase complex (Hara et al., 2006, J. Virol. 80:7789-7798). Theenzymology of the endonuclease within the context of intact viralribonucleoprotein particles (RNPs) has been extensively studied.

It has been found recently that, contrary to the general opinion in thefield, the endonucleolytic activity resides exclusively within the PAsubunit of the RNA-dependent RNA polymerase of the Influenza A H3N2virus (Dias et al., 2009).

The present inventors have now achieved to structurally characterize thePA domain of the Influenza A 2009 pandemic H1N1 virus by X-raycrystallography and identified the endonucleolytic active center withinsaid domain. The present inventors surprisingly found that polypeptidefragments of the PA subunit of said virus readily crystallized and that,thus, said polypeptide fragments are very suitable to study theendonucleolytic activity of the RNA-dependent RNA polymerase of theInfluenza A 2009 pandemic H1N1 virus in the context of said polypeptidefragments in order to simplify the development of new anti-viralcompounds targeting the endonuclease activity of said viral polymeraseas well as to optimize previously identified compounds.

The achievement of the present inventors to recombinantly produce PApolypeptide fragments possessing the endonucleolytic activity of theRNA-dependent RNA polymerase of the Influenza A 2009 pandemic H1N1 virusallows for performing in vitro high-throughput screening for inhibitorsof a functional site on said viral polymerase using easily obtainablematerial from a straightforward expression system. Furthermore, thestructural data of the endonucleolytic PA H1N1 polypeptide fragment aswell as of the enzymatically active center therein allows for directeddesign of inhibitors and in silico screening for potentially therapeuticcompounds.

The present inventors further managed, for the first time, theco-crystallization of a PA polypeptide fragment and of a variant thereofof the PA subunit of Influenza A 2009 pandemic H1N1 virus with a boundinhibitor and found that, thus, the development of new anti-viralcompounds targeting the endonuclease activity of the RNA-dependent RNApolymerase of the Influenza A 2009 pandemic H1N1 virus can be improved.Particularly, the co-crystallization data show, for the first time, indetail which amino acids comprised in the active site of a PApolypeptide fragment of the PA subunit of Influenza A 2009 pandemic H1N1virus are especially involved in compound binding. This new knowledgeallows the optimized design of modifications to existing inhibitors inorder to improve their potency or the design and optimization of novelinhibitors that effectively block endonuclease activity.

It is an object of the present invention to provide (i) high resolutionstructural data of the endonucleolytic amino-terminal domain of theviral polymerase H1N1 PA subunit by X-ray crystallography, (ii) highresolution structural data of the endonucleolytic amino-terminal domainof the viral polymerase H1N1 PA subunit co-crystallized with a knowninhibitor by X-ray crystallography, (iii) computational as well as invitro methods, preferably in a high-throughput setting, for identifyingcompounds that can modulate, preferably inhibit, the endonucleaseactivity of the viral polymerase of the Influenza A 2009 pandemic H1N1virus, preferably by blocking the endonucleolytic active site within theH1N1 PA subunit, and (iv) pharmacological compositions comprising suchcompounds for the treatment of infectious diseases caused by virusesusing the cap snatching mechanism for synthesis of viral mRNA.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a polypeptidefragment comprising an amino-terminal fragment of the PA subunit of aviral RNA-dependent RNA polymerase possessing endonuclease activity,wherein said PA subunit is from Influenza A pandemic 2009 H1N1 virusaccording to SEQ ID NO: 2 or is a variant thereof, wherein said variantcomprises the amino acid serine at an amino acid position 186 accordingto SEQ ID NO: 2 or at an amino acid position corresponding thereto.

In a further aspect, the present invention relates to an isolatedpolynucleotide coding for an isolated polypeptide fragment according tothe present invention.

In a further aspect, the present invention relates to a recombinantvector comprising the isolated polynucleotide according to the presentinvention.

In a further aspect, the present invention relates to a recombinant hostcell comprising the isolated polynucleotide according to the presentinvention or the recombinant vector according to the present invention.

In a further aspect, the present invention relates to a method foridentifying compounds, which modulate the endonuclease activity of thePA subunit of a RNA-dependent RNA polymerase from Influenza A 2009pandemic H1N1 virus or a variant thereof comprising the steps of:

-   (a) constructing a computer model of the active site defined by (i)    the structure coordinates of the polypeptide fragment according to    the present invention as shown in FIG. 1, (ii) the structure    coordinates of the polypeptide fragment according to the present    invention as shown in FIG. 2, (iii) the structure coordinates of the    polypeptide fragment according to the present invention as shown in    FIG. 3, (iv) the structure coordinates of the polypeptide fragment    according to the present invention as shown in FIG. 4, (v) the    structure coordinates of the polypeptide fragment according to the    present invention as shown in FIG. 5, (vi) the structure coordinates    of the polypeptide fragment according to the present invention as    shown in FIG. 15, or (vii) the structure coordinates of the    polypeptide fragment according to the present invention as shown in    FIG. 16,-   (b) selecting a potential modulating compound by a method selected    from the group consisting of:

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound;

-   (c) employing computational means to perform a fitting program    operation between computer models of the said compound and the said    active site in order to provide an energy-minimized configuration of    the said compound in the active site; and-   (d) evaluating the results of said fitting operation to quantify the    association between the said compound and the active site model,    whereby evaluating the ability of said compound to associate with    the said active site.

In a further aspect, the present invention relates to a method foridentifying compounds, which modulate the endonuclease activity of thePA subunit of a RNA-dependent RNA polymerase from Influenza A 2009pandemic H1N1 virus or a variant thereof comprising the steps of:

-   (i) contacting the polypeptide fragment or variant thereof according    to the present invention or the recombinant host cell according to    the present invention with a test compound, and-   (ii) analyzing the ability of said test compound to modulate the    endonuclease activity of said PA subunit polypeptide fragment or    variant thereof.

In a further aspect, the present invention relates to a compound whichis able to modulate, preferably to inhibit, the endonuclease activity ofthe PA subunit polypeptide fragment or variant thereof according to thepresent invention. Preferably, said compound is identifiable by the (invitro) methods according to the present inventions.

In a further aspect, the present invention relates to a pharmaceuticalcomposition comprising the compound of the present invention or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable excipient(s) and/or carrier(s). Preferably,said pharmaceutical composition is producible according to the (invitro) methods of the present invention.

In a further aspect, the present invention relates to an antibodydirected against the active site of the PA subunit of the Influenza A2009 pandemic H1N1 virus according to SEQ ID NO: 2 or a variant thereof,wherein said variant comprises the amino acid serine at an amino acidposition 186 according to SEQ ID NO: 2 or at an amino acid positioncorresponding thereto.

In a further aspect, the present invention relates to the use of acompound according to the present invention, a pharmaceuticalcomposition according to the present invention, or an antibody accordingto the present invention for the manufacture of a medicament fortreating, ameliorating, or preventing disease conditions caused by viralinfections with viruses of the Orthomyxoviridae family, Bunyaviridaefamily and/or Arenviridae family, preferably caused by viral infectionswith Influenza A 2009 pandemic H1N1 virus.

In a further aspect, the present invention relates to the use of4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3),4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2),4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1), or[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG) for the manufacture of a medicament for treating, ameliorating,or preventing disease conditions caused by viral infections withInfluenza A 2009 pandemic H1N1 virus.

In a further aspect, the present invention relates to a compoundaccording to the present invention, a pharmaceutical compositionaccording to the present invention, or an antibody according to thepresent invention for treating, ameliorating, or preventing diseaseconditions caused by viral infections with viruses of theOrthomyxoviridae family, Bunyaviridae family and/or Arenviridae family,preferably caused by viral infections with Influenza A 2009 pandemicH1N1 virus.

In a further aspect, the present invention relates to4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3),4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2),4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1), or[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG) for treating, ameliorating, or preventing disease conditionscaused by viral infections with Influenza A 2009 pandemic H1N1 virus.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise. For example, if in a preferredembodiment the polypeptide fragment of the present invention correspondsto amino acids 1 to 198 of the amino acid sequence set forth in SEQ IDNO: 2 and in another preferred embodiment the PA polypeptide fragmentaccording to the present invention may be tagged with a peptide-tag thatis preferably cleavable from the PA polypeptide fragment, preferablyusing a TEV protease, it is a preferred embodiment of the invention thatthe polypeptide fragment corresponding to amino acids 1 to 198 of theamino acid sequence set forth in SEQ ID NO: 2 is tagged with apeptide-tag that is cleavable from the PA polypeptide using a TEVprotease.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kolb′, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

To practice the present invention, unless otherwise indicated,conventional methods of chemistry, biochemistry, and recombinant DNAtechniques are employed which are explained in the literature in thefield (cf., e.g., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps. Asused in this specification and the appended claims, the singular forms“a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Definitions

The term “polypeptide fragment” refers to a part of a protein which iscomposed of a single amino acid chain. The term “protein” comprisespolypeptide fragments that resume a secondary and tertiary structure andadditionally refers to proteins that are made up of several amino acidchains, i.e., several subunits, forming quaternary structures. The term“peptide” refers to short amino acid chains of up to 50 amino acids thatdo not necessarily assume secondary or tertiary structures. A “peptoid”is a peptidomimetic that results from the oligomeric assembly ofN-substituted glycines.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues occupy an analogous position in the polypeptidestructures. As is well known in the art, analogous positions in two ormore polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW (seewebsite at ebi.ac.uk/clustalw) or Align (see website atebi.ac.uk/emboss/align/index.html) using standard settings, preferablyfor Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend0.5. Those skilled in the art understand that it may be necessary tointroduce gaps in either sequence to produce a satisfactory alignment.Residues in two or more PA subunits are said to “correspond” if theresidues are aligned in the best sequence alignment. The “best sequencealignment” between two polypeptides is defined as the alignment thatproduces the largest number of aligned identical residues. The “regionof best sequence alignment” ends and, thus, determines the metes andbounds of the length of the comparison sequence for the purpose of thedetermination of the similarity score, if the sequence similarity,preferably identity, between two aligned sequences drops to less than30%, preferably less than 20%, more preferably less than 10% over alength of 10, 20 or 30 amino acids.

The present invention relates to Influenza A 2009 pandemic H1N1 virusRNA-dependent RNA polymerase PA subunit fragments possessingendonuclease activity. The term “RNA-dependent RNA polymerase PAsubunit” refers to the PA subunit of Influenza A 2009 pandemic H1N1virus having an amino acid sequence as set forth in SEQ ID NO: 2. Theterm “RNA-dependent RNA polymerase PA subunit variant” refers to a PAsubunit variant of Influenza A 2009 pandemic H1N1 virus which comprisesthe amino acid serine at an amino acid position 186 according to SEQ IDNO: 2 or at an amino acid position corresponding thereto and preferablyhas at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequencesimilarity, preferably sequence identity over the entire length of thefragment using the best sequence alignment and/or over the region of thebest sequence alignment, wherein the best sequence alignment isobtainable with art known tools, e.g., Align, using standard settings,preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend0.5, with the amino acid sequence set forth in SEQ ID NO: 2. It ispreferred that when a PA subunit variant is aligned with a PA subunitaccording to SEQ ID NO: 2 that the alignment will be over the entirelength of the two polypeptides and, thus, that the alignment score willbe determined on this basis. It is, however, possible that the PAsubunit variant may comprise C-terminal/N-terminal or internal deletionsor additions, e.g., through N- or C-terminal fusions. In this case, onlythe best aligned region is used for the assessment of similarity andidentity, respectively. Preferably, fragments derived from thesevariants show the indicated similarity and identity, respectively,preferably within the region required for endonuclease activity.Accordingly, any alignment between SEQ ID NO: 2 and a PA subunit variantshould preferably comprise the endonuclease active site. Thus, the abovesequence similarity and identity to SEQ ID NO: 2 occurs at least over alength of 80, 90, 100, 110, 120, 130, 140, 150, 160, 165, 170, 180, 190,200, 210, 220, 230, 240, 250, 300 or more amino acids, preferablycomprising the endonuclease active site.

The polypeptide fragments of the present invention are, thus, based onInfluenza A 2009 pandemic H1N1 virus RNA-dependent RNA polymerase PAsubunit or variants thereof as defined above. Accordingly, in thefollowing specification, the terms “polypeptide fragment(s)” and “PApolypeptide fragment(s)” always comprise fragments derived both from thePA protein as set out in SEQ ID NO: 2 and from PA protein variantsthereof, as set out above, possessing endonuclease activity.

However, the specification also uses the terms “PA polypeptide fragmentvariants” or “PA fragment variants” to specifically refer to PApolypeptide fragments or PA fragments possessing endonuclease activitythat are derived from Influenza A 2009 pandemic H1N1 virus RNA-dependentRNA polymerase PA subunit variants. The PA polypeptide fragments of thepresent invention, thus, preferably comprise, essentially consist orconsist of sequences of the naturally occurring Influenza A 2009pandemic H1N1 virus PA subunit. It is, however, also envisioned that thePA polypeptide fragment variants comprise the amino acid serine at anamino acid position 186 according to SEQ ID NO: 2 or at an amino acidposition corresponding thereto and preferably further contain amino acidsubstitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore amino acid positions, and/or have at least 60%, 65%, 70%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence similarity, preferably sequence identityover the entire length of the fragment using the best sequence alignmentand/or over the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g., Align,using standard settings, preferably EMBOSS::needle, Matrix: Blosum62,Gap Open 10.0, Gap Extend 0.5, with the amino acid sequence set forth inSEQ ID NO: 2. It is understood that PA fragments of the presentinvention may comprise additional amino acids not derived from PA, like,e.g., tags, enzymes etc., such additional amino acids will not beconsidered in such an alignment, i.e., are excluded from the calculationof the alignment score. In a preferred embodiment, the above indicatedalignment score is obtained when aligning the sequence of the fragmentwith SEQ ID NO: 2 at least over a length of 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 165, 170, 180, or 190 amino acids, wherein thesequence of SEQ ID NO: 2 preferably comprises the endonuclease activesite.

In a preferred embodiment, the PA polypeptide fragment variants compriseat least the amino acid residues corresponding to amino acid residues 1to 190 of Influenza A 2009 pandemic H1N1 virus PA subunit according toSEQ ID NO: 2 or consist of amino acid residues 1 to 190 of Influenza A2009 pandemic H1N1 virus PA subunit according to SEQ ID NO: 2 and haveat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity, preferablysequence identity over the entire length of the fragment using the bestsequence alignment and/or over the region of the best sequencealignment, wherein the best sequence alignment is obtainable with artknown tools, e.g., Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5, withamino acid residues 1 to 190 of the amino acid sequence set forth in SEQID NO: 2. More preferably, the PA polypeptide fragment variants compriseat least the amino acid residues corresponding to amino acid residues 1to 198 of Influenza A 2009 pandemic H1N1 virus PA subunit according toSEQ ID NO: 2 or consist of amino acid residues 1 to 198 of Influenza A2009 pandemic H1N1 virus PA subunit according to SEQ ID NO: 2 and haveat least 70%, more preferably 75%, more preferably 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% sequence similarity, preferably sequence identity over theentire length of the fragment using the best sequence alignment and/orover the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g., Align,using standard settings, preferably EMBOSS::needle, Matrix: Blosum62,Gap Open 10.0, Gap Extend 0.5, with the amino acid residues 1 to 198 ofthe amino acid sequence set forth in SEQ ID NO: 2. It is also preferredthat the PA polypeptide fragment variants comprise at least the aminoacid residues corresponding to amino acid residues 1 to 200 of InfluenzaA 2009 pandemic H1N1 virus PA subunit according to SEQ ID NO: 2 orconsist of amino acid residues 1 to 200 of Influenza A 2009 pandemicH1N1 virus PA subunit according to SEQ ID NO: 2 and have at least 60%,more preferably 65%, more preferably 70%, more preferably 75%, morepreferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity, preferablysequence identity over the entire length of the fragment using the bestsequence alignment and/or over the region of the best sequencealignment, wherein the best sequence alignment is obtainable with artknown tools, e.g., Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5, withamino acid residues 1 to 200 of the amino acid sequence set forth in SEQID NO: 2. It should be noted that all of the above mentioned preferredPA polypeptide fragment variants comprise the amino acid serine at anamino acid position 186 according to SEQ ID NO: 2 or at an amino acidposition corresponding thereto.

In the context of the present invention, the term “PA-Nter” refers to apolypeptide fragment which consists of amino acid residues 1 to 198 ofthe amino acid sequence as set forth in SEQ ID NO: 2 (PA H1N1 1 to 198)and optionally of an additional amino-terminal linker, i.e. MGSGMA (SEQID NO: 3). In the context of the present invention, the term “PA-Ntermutant” refers to a polypeptide fragment (variant) which consists ofamino acid residues 1 to 198 of the amino acid sequence as set forth inSEQ ID NO: 2 with amino acids 52 to 64 replaced by the amino acidglycine (PA H1N1 1 to 198 Δ52-64: Gly) and optionally of an additionalamino-terminal linker, i.e. MGSGMA (SEQ ID NO: 3).

The term “sequence similarity” means that amino acids at the sameposition of the best sequence alignment are identical or similar,preferably identical. “Similar amino acids” possess similarcharacteristics, such as polarity, solubility, hydrophilicity,hydrophobicity, charge, or size. Similar amino acids are preferablyleucine, isoleucine, and valine; phenylalanine, tryptophan, andtyrosine; lysine, arginine, and histidine; glutamic acid and asparticacid; glycine, alanine, and serine; threonine, asparagine, glutamine,and methionine. The skilled person is well aware of sequence similaritysearching tools, e.g., available on the World Wide Web (see website atebi.ac.uk/Tools.similarity.html).

The term “soluble”, as used herein, refers to a polypeptide fragmentwhich remains in the supernatant after centrifugation for 30 min at100,000×g in an aqueous buffer under physiologically isotonicconditions, for example, 0.14 M sodium chloride or sucrose, at a proteinconcentration of at least 200 μg/ml, preferably of at least 500 μg/ml,preferably of at least 1 mg/ml, more preferably of at least 2 mg/ml,even more preferably of at least 3 mg/ml, even more preferably of atleast 4 mg/ml, most preferably of at least 5 mg/ml in the absence ofdenaturants such as guanidine or urea in effective concentrations. Aprotein fragment that is tested for its solubility is preferablyexpressed in one of the cellular expression systems indicated below.

The term “purified” in reference to a polypeptide, does not requireabsolute purity such as a homogenous preparation, rather it representsan indication that the polypeptide is relatively purer than in thenatural environment. Generally, a purified polypeptide is substantiallyfree of other proteins, lipids, carbohydrates, or other materials withwhich it is naturally associated, preferably at a functionallysignificant level, for example, at least 85% pure, more preferably atleast 90% or 95% pure, most preferably at least 99% pure. The expression“purified to an extent to be suitable for crystallization” refers to apolypeptide that is 85% to 100%, preferably 90% to 100%, more preferably95% to 100% or 98% to 100% pure and can be concentrated to higher than 3mg/ml, preferably higher than 10 mg/ml, more preferably higher than 18mg/ml without precipitation. A skilled artisan can purify a polypeptideusing standard techniques for protein purification. A substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

The term “associate” as used in the context of identifying compoundswith the methods of the present invention refers to a condition ofproximity between a moiety (i.e., chemical entity or compound orportions or fragments thereof), and an endonuclease active site of thePA subunit. The association may be non-covalent, i.e., where thejuxtaposition is energetically favored by, for example,hydrogen-bonding, van der Waals, electrostatic, or hydrophobicinteractions, or it may be covalent.

The term “endonuclease activity” or “endonucleolytic activity” refers toan enzymatic activity which results in the cleavage of thephosphodiester bond within a polynucleotide chain. In the context of thepresent invention, the polypeptide fragments possess an endonucleolyticactivity, which is preferably not selective for the polynucleotide type,i.e., the polypeptide fragments according to the present inventionpreferably exhibit endonucleolytic activity for DNA and RNA, preferablyfor single stranded DNA (ssDNA) or single stranded RNA (ssRNA). In thiscontext, “Single stranded” means that a stretch of preferably at least 3nucleotides, preferably at least 5 nucleotides, more preferably at least10 nucleotides within the polynucleotide chain are single stranded,i.e., not base paired to another nucleotide. Preferably, theendonucleolytic activity of the polypeptide fragments according to thepresent invention is not dependent on recognition sites, i.e., specificnucleotide sequences, but results in unspecific cleavage ofpolynucleotide chains. For example, the skilled person may test forendonucleolytic activity of polypeptide fragments according to thepresent invention by incubating RNA or DNA substrates such as panhandleRNA or a linear or circular single stranded DNA, with or without therespective polypeptide fragment, for example, at 37° C. for a certainperiod of time such as for 5, 10, 20, 40, 60, or 80 minutes, and testfor the integrity of the polynucleotides, for example, by gelelectrophoresis.

The term “nucleotide” as used herein refers to a compound consisting ofa purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e.g., as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992) and further include,but are not limited to, synthetic nucleosides having modified basemoieties and/or modified sugar moieties, e.g., described generally byScheit, Nucleotide Analogs (John Wiley, N.Y., 1980).

The term “isolated polynucleotide” refers to polynucleotides that were(i) isolated from their natural environment, (ii) amplified bypolymerase chain reaction, or (iii) wholly or partially synthesized, andmeans a single or double-stranded polymer of deoxyribonucleotide orribonucleotide bases and includes DNA and RNA molecules, both sense andanti-sense strands. The term comprises cDNA, cRNA, genomic DNA, andrecombinant DNA. A polynucleotide may consist of an entire gene, or aportion thereof.

The term “recombinant vector” as used herein includes any vectors knownto the skilled person including plasmid vectors, cosmid vectors, phagevectors such as lambda phage, viral vectors such as adenoviral orbaculoviral vectors, or artificial chromosome vectors such as bacterialartificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1artificial chromosomes (PAC). Said vectors include expression as well ascloning vectors. Expression vectors comprise plasmids as well as viralvectors and generally contain a desired coding sequence and appropriateDNA sequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments.

“Recombinant host cell”, as used herein, refers to a host cell thatcomprises a polynucleotide that codes for a polypeptide fragment ofinterest, i.e., the Influenz A pandemic H1N1 PA polypeptide fragment orvariants thereof according to the invention. This polynucleotide may befound inside the host cell (i) freely dispersed as such, (ii)incorporated in a recombinant vector, or (iii) integrated into the hostcell genome or mitochondrial DNA. The recombinant cell can be used forexpression of a polynucleotide of interest or for amplification of thepolynucleotide or the recombinant vector of the invention. The term“recombinant host cell” includes the progeny of the original cell whichhas been transformed, transfected, or infected with the polynucleotideor the recombinant vector of the invention. A recombinant host cell maybe a bacterial cell such as an E. coli cell, a yeast cell such asSaccharomyces cerevisiae or Pichia pastoris, a plant cell, an insectcell such as SF9 or High Five cells, or a mammalian cell. Preferredexamples of mammalian cells are Chinese hamster ovary (CHO) cells, greenAfrican monkey kidney (COS) cells, human embryonic kidney (HEK293)cells, HELA cells, and the like.

As used herein, the term “crystal” or “crystalline” means a structure(such as a three-dimensional solid aggregate) in which the plane facesintersect at definite angles and in which there is a regular structure(such as internal structure) of the constituent chemical species. Theterm “crystal” can include any one of: a solid physical crystal formsuch as an experimentally prepared crystal, a crystal structurederivable from the crystal (including secondary and/or tertiary and/orquaternary structural elements), a 2D and/or 3D model based on thecrystal structure, a representation thereof such as a schematicrepresentation thereof or a diagrammatic representation thereof, or adata set thereof for a computer. In one aspect, the crystal is usable inX-ray crystallography techniques. Here, the crystals used can withstandexposure to X-ray beams and are used to produce diffraction pattern datanecessary to solve the X-ray crystallographic structure. A crystal maybe characterized as being capable of diffracting X-rays in a patterndefined by one of the crystal forms depicted in T. L. Blundell and L. N.Johnson, “Protein Crystallography”, Academic Press, New York (1976). Theterm “unit cell” refers to a basic cubic or parallelepiped shaped block.The entire volume of a crystal may be constructed by regular assembly ofsuch blocks. Each unit cell comprises a complete representation of theunit of pattern, the repetition of which builds up the crystal.

The term “space group” refers to the arrangement of symmetry elements ofa crystal. In a space group designation the capital letter indicates thelattice type and the other symbols represent symmetry operations thatcan be carried out on the contents of the asymmetric unit withoutchanging its appearance.

The term “structure coordinates” refers to a set of values that definethe position of one or more amino acid residues with reference to asystem of axes. The term refers to a data set that defines thethree-dimensional structure of a molecule or molecules (e.g., Cartesiancoordinates, temperature factors, and occupancies). Structuralcoordinates can be slightly modified and still render nearly identicalthree-dimensional structures. A measure of a unique set of structuralcoordinates is the root mean square deviation of the resultingstructure. Structural coordinates that render three-dimensionalstructures (in particular, a three-dimensional structure of anenzymatically active center) that deviate from one another by a rootmean square deviation of less than 3 Å, 2 Å, 1.5 Å, 1.0 Å, or 0.5 Å maybe viewed by a person of ordinary skill in the art as very similar.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a variant of the PA polypeptide fragment orthe enzymatically active center therein from the backbone of the PApolypeptide fragment or the enzymatically active center therein asdefined by the structure coordinates of the PA polypeptide fragmentPA-Nter according to FIG. 1.

As used herein, the term “constructing a computer model” includes thequantitative and qualitative analysis of molecular structure and/orfunction based on atomic structural information and interaction models.The term “modeling” includes conventional numeric-based moleculardynamic and energy minimization models, interactive computer graphicmodels, modified molecular mechanics models, distance geometry, andother structure-based constraint models.

The term “fitting program operation” refers to an operation thatutilizes the structure coordinates of a chemical entity, anenzymatically active center, a binding pocket, molecule or molecularcomplex, or portion thereof, to associate the chemical entity with theenzymatically active center, the binding pocket, molecule or molecularcomplex, or portion thereof. This may be achieved by positioning,rotating or translating the chemical entity in the enzymatically activecenter to match the shape and electrostatic complementarity of theenzymatically active center. Covalent interactions, non-covalentinteractions such as hydrogen bond, electrostatic, hydrophobic, van derWaals interactions, and non-complementary electrostatic interactionssuch as repulsive charge-charge, dipole-dipole and charge-dipoleinteractions may be optimized. Alternatively, one may minimize thedeformation energy of binding of the chemical entity to theenzymatically active center.

As used herein, the term “test compound” refers to an agent comprising acompound, molecule, or complex that is being tested for its ability toinhibit the endonucleolytic activity of the polypeptide fragment ofinterest, i.e., the PA polypeptide fragment of the invention or variantsthereof possessing endonucleolytic acitvity. Test compounds can be anyagents including, but not restricted to, peptides, peptoids,polypeptides, proteins (including antibodies), lipids, metals,nucleotides, nucleotide analogs, nucleosides, nucleic acids, smallorganic or inorganic molecules, chemical compounds, elements,saccharides, isotopes, carbohydrates, imaging agents, lipoproteins,glycoproteins, enzymes, analytical probes, polyamines, and combinationsand derivatives thereof. The term “small molecules” refers to moleculesthat have a molecular weight between 50 and about 2,500 Daltons,preferably in the range of 200-800 Daltons. In addition, a test compoundaccording to the present invention may optionally comprise a detectablelabel. Such labels include, but are not limited to, enzymatic labels,radioisotope or radioactive compounds or elements, fluorescent compoundsor metals, chemiluminescent compounds and bioluminescent compounds. Wellknown methods may be used for attaching such a detectable label to atest compound. The test compound of the invention may also comprisecomplex mixtures of substances, such as extracts containing naturalproducts, or the products of mixed combinatorial syntheses. These canalso be tested and the component that inhibits the endonucleolyticactivity of the target polypeptide fragment can be purified from themixture in a subsequent step. Test compounds can be derived or selectedfrom libraries of synthetic or natural compounds. For instance,synthetic compound libraries are commercially available from MaybridgeChemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (SanDiego, Calif.), or Aldrich (Milwaukee, Wis.). A natural compound libraryis, for example, available from TimTec LLC (Newark, Del.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal cell and tissue extracts can be used.Additionally, test compounds can be synthetically produced usingcombinatorial chemistry either as individual compounds or as mixtures. Acollection of compounds made using combinatorial chemistry is referredto herein as a combinatorial library.

In the context of the present invention, “a compound which modulates theendonucleolytic activity” may increase or decrease, preferably inhibitthe endonucleolytic activity of the PA subunit of the Influenza A 2009pandemic H1N1 virus or the Influenza A 2009 pandemic H1N1 virusRNA-dependent RNA polymerase or a variant thereof. Preferably, such acompound is specific for the endonucleolytic activity of the Influenza A2009 pandemic H1N1 PA subunit or variant thereof and does not modulate,preferably decrease the endonucleolytic activity of other endonucleases,in particular mammalian endonucleases.

The term “a compound which decreases the endonucleolytic activity” meansa compound which decreases the endonucleolytic activity of the PAsubunit of the Influenza A 2009 pandemic H1N1 virus or the Influenza A2009 pandemic H1N1 virus RNA-dependent RNA polymerase or a variantthereof by 50%, more preferably by 60%, even more preferably by 70%,even more preferably by 80%, even more preferably by 90%, and mostpreferably by 100% compared to the endonucleolytic activity of said PAsubunit or a variant thereof without said compound but with otherwisethe same reaction conditions, i.e., buffer conditions, reaction time andtemperature.

It is most preferred that the compound which decreases theendonucleolytic activity of the PA subunit of the Influenza A 2009pandemic H1N1 virus or a variant thereof inhibits said activity, i.e.,decreases said activity by at least 95%, preferably by 100% compared tothe activity without the compound. It is particularly preferred that thecompound that decreases or inhibits the endonucleolytic activity of thePA subunit of the Influenza A 2009 pandemic H1N1 virus or a variantthereof specifically decreases or inhibits the endonucleolytic activityof said PA subunit or a variant thereof but does not inhibit theendonucleolytic activity of other endonucleases such as RNase H orrestriction endonucleases to the same extent, preferably not at all. Forexample, the skilled person may set up the following samples with thesame buffer and reaction conditions as well as substrate andendonuclease concentrations: (1) substrate such as panhandle RNA,endonucleolytically active PA H1N1 polypeptide fragment or variantthereof, (2) substrate such as panhandle RNA, endonucleolytically activePA H1N1 polypeptide fragment or variant thereof, test compound, (3)substrate such as panhandle RNA, reference endonuclease such as RNAse H,(4) substrate such as panhandle RNA, reference nucleotide such as RNAseH, test compound. After incubation of the samples, the skilled personmay analyze the substrate, for example, by gel electrophoresis. Testcompounds which result in cleaved substrate in sample (4) and intactsubstrate in sample (2) are preferred.

The term “in a high-throughput setting” refers to high-throughputscreening assays and techniques of various types which are used toscreen libraries of test compounds for their ability to inhibit theendonuclease activity of the polypeptide fragment of interest.Typically, the high-throughput assays are performed in a multi-wellformat and include cell-free as well as cell-based assays.

The term “antibody” refers to both monoclonal and polyclonal antibodies,i.e., any immunoglobulin protein or portion thereof which is capable ofrecognizing an antigen or hapten, i.e., the Influenza A 2009 pandemicH1N1 PA polypeptide fragment possessing endonucleolytic activity or apeptide thereof. In a preferred embodiment, the antibody is capable ofbinding to the enzymatically (endonucleolytically) active center withinthe Influenza A 2009 pandemic H1N1 PA polypeptide fragment or variantthereof. Antigen-binding portions of the antibody may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. In some embodiments, antigen-binding portions includeFab, Fab′, F(ab′)₂, Fd, Fv, dAb, and complementarity determining region(CDR) fragments, single-chain antibodies (scFv), chimeric antibodiessuch as humanized antibodies, diabodies, and polypeptides that containat least a portion of an antibody that is sufficient to confer specificantigen binding to the polypeptide.

The term “pharmaceutically acceptable salt” refers to a salt of acompound identifiable by the methods of the present invention or acompound of the present invention. Suitable pharmaceutically acceptablesalts include acid addition salts which may, for example, be formed bymixing a solution of compounds of the present invention with a solutionof a pharmaceutically acceptable acid such as hydrochloric acid,sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid,benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoricacid. Furthermore, where the compound carries an acidic moiety, suitablepharmaceutically acceptable salts thereof may include alkali metal salts(e.g., sodium or potassium salts); alkaline earth metal salts (e.g.,calcium or magnesium salts); and salts formed with suitable organicligands (e.g., ammonium, quaternary ammonium and amine cations formedusing counteranions such as halide, hydroxide, carboxylate, sulfate,phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrativeexamples of pharmaceutically acceptable salts include, but are notlimited to, acetate, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, butyrate, calcium edetate, camphorate, camphorsulfonate,camsylate, carbonate, chloride, citrate, clavulanate,cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate,edetate, edisylate, estolate, esylate, ethanesulfonate, formate,fumarate, gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate,mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like(see, for example, S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19 (1977)).

The term “excipient” when used herein is intended to indicate allsubstances in a pharmaceutical formulation which are not activeingredients such as, e.g., carriers, binders, lubricants, thickeners,surface active agents, preservatives, emulsifiers, buffers, flavoringagents, or colorants.

The term “pharmaceutically acceptable carrier” includes, for example,magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.

DETAILED DESCRIPTION

The present inventors surprisingly found that a small independentlyfolded domain derived from the N-terminus of the PA subunit of InfluenzaA 2009 pandemic H1N1 virus RNA-dependent RNA polymerase exhibits thefunctional properties of the endonuclease reported for the trimericcomplex, although this activity was thought to be detectable only in thetrimeric complex. Moreover, the inventors found that this PA polypeptidefragment can easily be produced by recombinant means and, thus, issuitable for in vitro studies on the endonucleolytic activity and andits modulation. The present inventors surprisingly found that the domainderived from the N-terminus of the PA subunit of Influenza A 2009pandemic H1N1 virus RNA-dependent RNA polymerase readily crystallizedand that, thus, said domain is very dedicated for the identification,selection and design of new anti-viral compounds targeting theendonuclease activity of the Influenza A 2009 pandemic H1N1 polymeraseas well as for the optimization of previously identified compounds bycomputer methods.

It is one aspect of the present invention to provide a polypeptidefragment comprising an amino-terminal fragment of the PA subunit of aviral RNA-dependent RNA polymerase possessing endonuclease activity,wherein said PA subunit is from Influenza A 2009 pandemic H1N1 virusaccording to SEQ ID NO: 2 or is a variant thereof, wherein said variantcomprises the amino acid serine at an amino acid position 186 accordingto SEQ ID NO: 2 or at an amino acid position corresponding thereto.

In a preferred embodiment of the present invention, said variantcomprises the amino acids 1 to 198 of the amino acid sequence set forthin SEQ ID NO: 2 with amino acids 52 to 64 replaced by the amino acidglycine (SEQ ID NO:14). In a further preferred embodiment of the presentinvention, said variant comprises the amino acids 1 to 198 of the aminoacid sequence set forth in SEQ ID NO: 2 with amino acids 52 to 72replaced by the amino acid glycine (SEQ ID NO:15). In another furtherpreferred embodiment of the present invention, said variant comprisesthe amino acids 1 to 198 of the amino acid sequence set forth in SEQ IDNO: 2 with amino acids 52 to 69 replaced by the amino acid glycine (SEQID NO:16).

It is preferred that the polypeptide fragment according to the presentinvention is soluble, preferably in an aqueous solution. The minimallength of the polypeptide fragment of the present invention isdetermined by its ability to cleave polynucleotide chains such aspanhandle RNA or single stranded DNA, i.e., the minimal length of thepolypeptide is determined by its endonucleolytic activity. Preferably,the endonuclease activity is not dependent on the polynucleotide type,and thus, may be exerted on DNA and RNA, preferably on single strandedDNA and RNA. Preferably, the endonuclease activity is not dependent onspecific recognition sites within the substrate polynucleotide.

In a preferred embodiment, the polypeptide fragment according to thepresent invention is suitable for crystallization, i.e., preferably thepolypeptide fragment is crystallizable. Preferably, the crystalsobtainable from the polypeptide fragment according to the invention aresuitable for structure determination of the polypeptide fragment usingX-ray crystallography. Preferably, said crystals are greater than 25micron cubes and preferably are radiation stable enough to permit morethan 85% diffraction data completeness at resolution of preferably 3.5 Åor better to be collected upon exposure to monochromatic X-rays.

In one embodiment, the polypeptide fragment is crystallizable using (i)an aqueous protein solution, i.e. the crystallization solution, with aprotein concentration of 5 to 20 mg/ml, e.g. of 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/ml, preferably of8 to 15 mg/ml, most preferably of 10 to 15 mg/ml in a buffer system suchas HEPES or Tris-HCl at concentrations ranging from 10 mM to 3 M,preferably 10 mM to 2 M, more preferably 20 mM to 1 M, at pH 3 to pH 9,preferably pH 4 to pH 9, more preferably pH 7 to pH 9, and (ii) aprecipitant/reservoir solution comprising one or more substances such assodium formate, ammonium sulphate, lithium sulphate, magnesium acetate,manganese acetate, sodium chloride, glycerol or ethylene glycol.

Optionally, the protein solution may contain one or more salts such asmonovalent salts, e.g., NaCl, KCl, or LiCl, preferably NaCl, atconcentrations ranging from 10 mM to 1 M, preferably 20 mM to 500 mM,more preferably 50 mM to 200 mM, and/or divalent salts, e.g., MnCl₂,CaCl₂, MgCl₂, ZnCl₂, or CoCl₂, preferably MgCl₂ and MnCl₂, atconcentrations ranging from 0.1 to 50 mM, preferably 0.5 to 25 mM, morepreferably 1 to 10 mM or 1 to 5 mM.

Preferably, the precipitant/reservoir solution comprises sodium formateat concentrations ranging from 0.5 to 2 M, preferably 1 to 1.8 M, abuffer system such as HEPES at concentrations ranging from 10 mM to 1 M,preferably 50 mM to 500 mM, more preferably 75 to 150 mM, at preferablypH 4 to 8, more preferably pH 5 to 7, and/or ethylene glycol atconcentrations ranging from 1% to 20%, preferably 2% to 8%, morepreferably 2 to 5%.

The PA polypeptide fragment or variant thereof is preferably 85% to 100%pure, more preferably 90% to 100% pure, even more preferably 95% to 100%pure in the crystallization solution. To produce crystals, the proteinsolution suitable for crystallization may be mixed with an equal volumeof the precipitant solution.

In a preferred embodiment, the crystallization medium comprises 0.05 to2 μl, preferably 0.8 to 1.2 μl, of protein solution suitable forcrystallization mixed with a similar, preferably equal volume ofprecipitant solution comprising 1.0 to 2.0 M sodium formate, 80 to 120mM HEPES pH 6.5 to pH 7.5, and 2 to 5% glycol or glycerol.

In another embodiment, the precipitant solution comprises, preferablyessentially consists of or consists of 1.6 M sodium formate, 0.1 M HEPESpH 7.0, and 5% glycol or glycerol, and the crystallization/proteinsolution comprises, preferably essentially consists or consists of 10 to15 mg/ml in 20 mM HEPES pH 7.5, 150 mM NaCl, 2.0 mM MnCl₂, and 2.0 mMMgCl₂.

In another embodiment, the PA polypeptide fragment is co-crystallizablewith a compound, preferably with a compound that modulates, preferablyinhibits, the endonuclease activity of the PA polypeptide fragment,preferably with a compound according to Tables 1 or 2, using (i) anaqueous protein solution with a concentration of the PA polypeptidefragment of 5 to 20 mg/ml, e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5,17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/ml, preferably of 8 to 15 mg/ml,most preferably of 10 to 15 mg/ml in a buffer system such as HEPES orTris-HCl at concentrations ranging from 10 mM to 3 M, preferably 10 mMto 2 M, more preferably 20 mM to 1 M, at pH 3 to pH 9, preferably pH 4to pH 9, more preferably pH 7 to pH 9, and (ii) a precipitant/reservoirsolution comprising one or more substances such as sodium formate,ammonium sulphate, lithium sulphate, magnesium acetate, manganeseacetate, sodium chloride, ethylene glycol, glycerol, or PEG. Forco-crystallization, a compound, preferably a compound that modulates,e.g. inhibits, the endonuclease activity of the PA polypeptide fragment,preferably a compound according to Tables 1 or 2, is added to theaqueous protein solution to a final concentration of between 0.5 and 5mM, preferably of between 1.5 and 5 mM, i.e. 0.5, 1, 1.5, 2, 2.5, 3, 4.5or 5 mM.

Optionally, the protein solution may contain one or more salts such asmonovalent salts, e.g., NaCl, KCl, or LiCl, preferably NaCl, atconcentrations ranging from 10 mM to 1 M, preferably 20 mM to 500 mM,more preferably 50 mM to 200 mM, and/or divalent salts, e.g., MnCl₂,CaCl₂, MgCl₂, ZnCl₂, or CoCl₂, preferably MgCl₂ and MnCl₂, atconcentrations ranging from 0.1 to 50 mM, preferably 0.5 to 25 mM, morepreferably 1 to 10 mM or 1 to 5 mM.

Preferably, the precipitant/reservoir solution comprises ammoniumsulphate at concentrations ranging from 0.1 to 2.5 M, preferably 0.1 to2.0 M, a buffer system such as Bis-Tris at concentrations ranging from10 mM to 1 M, preferably 50 mM to 500 mM, more preferably 75 to 150 mM,at preferably pH 4 to 7, more preferably pH 5 to 6, and/or PEG such asPEG 3350 at concentrations ranging from 1% to 30%, preferably 15% to30%, more preferably 20 to 25%.

The PA polypeptide fragment or variant thereof is preferably 85% to 100%pure, more preferably 90% to 100% pure, even more preferably 95% to 100%pure in the protein solution. For co-crystallization, the aqueousprotein solution comprising the PA polypeptide fragment or variantthereof and the compound may be mixed with an equal volume of theprecipitant solution.

In a preferred embodiment, the protein solution comprises, preferablyessentially consists or consists of 10 to 15 mg/ml PA polypeptidefragment in 20 mM HEPES pH 7.5, 150 mM NaCl, 2.0 mM MnCl₂, and 2.0 mMMgCl₂ and4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3) in a final concentration of 1.5 mM, and the reservoirsolution/precipitant solution comprises, preferably essentially consistsof or consists of 2.0 M ammonium sulphate and 0.1M Bis-Tris pH5.5 (seealso Table 1).

In another preferred embodiment, the protein solution comprises,preferably essentially consists or consists of 10 to 15 mg/ml PApolypeptide fragment in 20 mM HEPES pH 7.5, 150 mM NaCl, 2.0 mM MnCl₂,and 2.0 mM MgCl₂ and4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2) in a final concentration of 1.5 mM, preferably soakedinto crystals initially grown with ribo-Uridine Monophosphate (rUMP),and the reservoir solution/precipitant solution comprises, preferablyessentially consists of or consists of 0.1 M ammonium sulphate, 0.1 MBis-Tris pH5.5 and 25% (w/v) PEG 3350 (see also Table 1).

In a further preferred embodiment, the protein solution comprises,preferably essentially consists or consists of 10 to 15 mg/ml PApolypeptide fragment in 20 mM HEPES pH 7.5, 150 mM NaCl, 2.0 mM MnCl₂,and 2.0 mM MgCl₂ and ribo-Uridine Monophosphate (rUMP) in a finalconcentration of 5 mM, and the reservoir solution/precipitant solutioncomprises, preferably essentially consists of or consists of 0.1 Mammonium sulphate, 0.1 M Bis-Tris pH5.5 and 25% (w/v) PEG 3350 (see alsoTable 1).

Other preferred reservoir solutions/precipitant solutions for theco-crystallization of the PA polypeptide fragment or variant thereof,e.g. with4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1) or with[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG), can further be taken from Table 1.

Crystals can be grown by any method known to the person skilled in theart including, but not limited to, hanging and sitting drop techniques,sandwich-drop, dialysis, and microbatch or microtube batch devices. Itwould be readily apparent to one of skill in the art to vary thecrystallization conditions disclosed above to identify othercrystallization conditions that would produce crystals of PA polypeptidefragments of the inventions or variants thereof alone or in complex witha compound. Such variations include, but are not limited to, adjustingpH, protein concentration and/or crystallization temperature, changingthe identity or concentration of salt and/or precipitant used, using adifferent method for crystallization, or introducing additives such asdetergents (e.g., TWEEN 20 (monolaurate), LDOA, Brij 30 (4 laurylether)), sugars (e.g., glucose, maltose), organic compounds (e.g.,dioxane, dimethylformamide), lanthanide ions, or poly-ionic compoundsthat aid in crystallizations. High throughput crystallization assays mayalso be used to assist in finding or optimizing the crystallizationcondition.

Microseeding may be used to increase the size and quality of crystals.In brief, micro-crystals are crushed to yield a stock seed solution. Thestock seed solution is diluted in series. Using a needle, glass rod orstrand of hair, a small sample from each diluted solution is added to aset of equilibrated drops containing a protein concentration equal to orless than a concentration needed to create crystals without the presenceof seeds. The aim is to end up with a single seed crystal that will actto nucleate crystal growth in the drop.

The manner of obtaining the structure coordinates as shown in FIGS. 1 to5, interpretation of the coordinates and their utility in understandingthe protein structure, as described herein, are commonly understood bythe skilled person and by reference to standard texts such as J. Drenth,“Principles of protein X-ray crystallography”, 2^(nd) Ed., SpringerAdvanced Texts in Chemistry, New York (1999); and G. E. Schulz and R. H.Schirmer, “Principles of Protein Structure”, Springer Verlag, New York(1985). For example, X-ray diffraction data is first acquired, oftenusing cryoprotected (e.g., with 20% to 30% glycerol) crystals frozen to100 K, e.g., using a beamline at a synchrotron facility or a rotatinganode as an X-ray source. Then, the phase problem is solved by agenerally known method, e.g., multiwavelength anomalous diffraction(MAD), multiple isomorphous replacement (MIR), single wavelengthanomalous diffraction (SAD), or molecular replacement (MR). Thesub-structure may be solved using SHELXD (Schneider and Sheldrick, 2002,Acta Crystallogr. D. Biol. Crystallogr. (Pt 10 Pt 2), 1772-1779), phasescalculated with SHARP (Vonrhein et al., 2006, Methods Mol. Biol.364:215-30), and improved with solvent flattening andnon-crystallographic symmetry averaging, e.g., with RESOLVE(Terwilliger, 2000, Acta Cryst. D. Biol. Crystallogr. 56:965-972). Modelautobuilding can be done, e.g., with ARP/wARP (Perrakis et al., 1999,Nat. Struct. Biol. 6:458-63) and refinement with, e.g. REFMAC(Murshudov, 1997, Acta Crystallogr. D. Biol. Crystallogr. 53: 240-255).

Preferably, the amino terminal PA fragment comprised within thepolypeptide fragment according to the present invention corresponds to,preferably essentially consists or consists of, at least amino acids 1to 190, preferably amino acids 1 to 198, preferably amino acids 1 to200, of the PA subunit of the RNA-dependent RNA polymerase of InfluenzaA 2009 pandemic H1N1 virus according to SEQ ID NO: 2 or variantsthereof.

In a preferred embodiment, the polypeptide fragment according to thepresent invention is purified to an extent to be suitable forcrystallization, preferably it is 85% to 100%, more preferably 90% to100%, most preferably 95% to 100% pure. In another preferred embodiment,the polypeptide fragment according to the present invention is purifiedto an extent to be suitable for co-crystallization, preferably it is 85%to 100%, more preferably 90% to 100%, most preferably 95% to 100% pure.

In another embodiment, the polypeptide fragment according to the presentinvention is capable of binding to divalent cations. Preferably, thepolypeptide fragment according to the present invention is bound to oneor more divalent cation(s), preferably it is bound to two divalentcations. In this context, the divalent cation is preferably selectedform the group consisting of manganese, cobalt, calcium, magnesium, andzinc, and is more preferably manganese or cobalt, most preferablymanganese and/or magnesium. Thus, in a preferred embodiment, thepolypeptide fragment of the present invention is present in complex with(i) two manganese cations, (ii) two magnesium cations, or (iii) onemanganese and one magnesium cation. In a preferred embodiment, thedivalent cations are coordinated by amino acids corresponding to aminoacids Glu80 and Asp108 (second cation) and amino acids corresponding toamino acids His41, Asp108, Ile120 and Glu119 (first cation) as set forthin SEQ ID NO: 2. In preferred embodiment, the divalent cation ismanganese for site 1 and manganese or magnesium for site 2, i.e.manganese for site 1 and magnesium for site 2 (see FIG. 6) or manganesefor site 1 and manganese for site 2 (see FIGS. 7 to 10, 17 and 18)

In a further embodiment, the polypeptide fragment according to thepresent invention is a polypeptide fragment, wherein the N-terminus isidentical to or corresponds to amino acid position 15 or lower, e.g., atposition 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and theC-terminus is identical to or corresponds to an amino acid at a positionselected from positions 186 to 200, e.g., 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, or 200 of the amino acidsequence of the PA subunit according to SEQ ID NO: 2, preferably theC-terminus is identical to or corresponds to an amino acid at a positionselected from 190 to 200, more preferably 190 to 198 of the amino acidsequence of the PA subunit according to SEQ ID NO: 2, or is a variantthereof, wherein said variant comprises the amino acid serine at anamino acid position 186 according to SEQ ID NO: 2 or at an amino acidposition corresponding thereto and retains the endonuclease activity.

In a preferred embodiment of the present invention, said variantcomprises SEQ ID NO:14 (the amino acids 1 to 198 of the amino acidsequence set forth in SEQ ID NO: 2 with amino acids 52 to 64 replaced bythe amino acid glycine). In a more preferred embodiment of the presentinvention, said variant consists of SEQ ID NO:14 (the amino acids 1 to198 of the amino acid sequence set forth in SEQ ID NO: 2 with aminoacids 52 to 64 replaced by the amino acid glycine) and optionally of anamino-terminal linker having the amino acid sequence MGSGMA (SEQ ID NO:3).

In a further preferred embodiment of the present invention, said variantcomprises SEQ ID NO:15 (the amino acids 1 to 198 of the amino acidsequence set forth in SEQ ID NO: 2 with amino acids 52 to 72 replaced bythe amino acid glycine). In a more preferred embodiment of the presentinvention, said variant consists of SEQ ID NO:15 (the amino acids 1 to198 of the amino acid sequence set forth in SEQ ID NO: 2 with aminoacids 52 to 72 replaced by the amino acid glycine) and optionally of anamino-terminal linker having the amino acid sequence MGSGMA (SEQ ID NO:3).

In another further preferred embodiment of the present invention, saidvariant comprises SEQ ID NO:16 (the amino acids 1 to 198 of the aminoacid sequence set forth in SEQ ID NO: 2 with amino acids 52 to 69replaced by the amino acid glycine). In a more preferred embodiment ofthe present invention, said variant consists of SEQ ID NO:16 (the aminoacids 1 to 198 of the amino acid sequence set forth in SEQ ID NO: 2 withamino acids 52 to 69 replaced by the amino acid glycine) and optionallyof an amino-terminal linker having the amino acid sequence MGSGMA (SEQID NO: 3).

Preferably, said polypeptide fragment has or corresponds to an aminoacid sequence selected from the group of amino acid sequences consistingof amino acids 5 to 190, 10 to 190, 15 to 190, 20 to 190, 5 to 198, 10to 198, 15 to 198, 20 to 198 of the amino acid sequence set forth in SEQID NO: 2 and variants thereof, wherein said variants comprises the aminoacid serine at an amino acid position 186 according to SEQ ID NO: 2 orat an amino acid position corresponding thereto and retain theendonuclease activity.

In a further embodiment, the polypeptide fragment (variant) according tothe present invention

-   (a) consists of SEQ ID NO:13 (amino acids 1 to 198 of the amino acid    sequence set forth in SEQ ID NO: 2) and optionally of an    amino-terminal linker having the amino acid sequence MGSGMA (SEQ ID    NO: 3) and has the structure defined by (i) the structure    coordinates as shown in FIG. 1, (ii) the structure coordinates as    shown in FIG. 2, (iii) the structure coordinates as shown in FIG.    3, (iv) the structure coordinates as shown in FIG. 4, or (v) the    structure coordinates as shown in FIG. 5, or-   (b) consists of SEQ ID NO:14 (amino acids 1 to 198 of the amino acid    sequence set forth in SEQ ID NO: 2 with amino acids 52 to 64    replaced by the amino acid glycine) and optionally of an    amino-terminal linker having the amino acid sequence MGSGMA (SEQ ID    NO: 3) and has the structure defined by (vi) the structure    coordinates as shown in FIG. 15, or (vii) the structure coordinates    as shown in FIG. 16.

It is preferred that said polypeptide fragment according to the presentinvention has the crystal structure defined by the structure coordinatesas shown in FIG. 1 without co-crystallization with a compound (nativestructure).

It is further preferred that said polypeptide fragment (variant)according to the present invention has the crystal structure defined bythe structure coordinates as shown in FIG. 2 to 5, 15 or 16 afterco-crystallization with a compound, preferably with a compound thatmodulates, preferably inhibits, the endonuclease activity of saidpolypeptide fragment. It is particularly preferred that said polypeptidefragment (variant) has the structure defined by the structurecoordinates as shown in FIG. 2 to 5, 15 or 16 after co-crystallizationwith a compound according to Tables 1 or 2, i.e. the structure definedby the structure coordinates as shown in FIGS. 2 and 3 afterco-crystallization with4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3), the structure defined by the structure coordinates asshown in FIG. 4 after co-crystallization with4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid, (EMBL-R05-2), the structure defined by the structure coordinatesas shown in FIG. 5 after co-crystallization with ribo-uridinemonophosphate (rUMP), the structure defined by the structure coordinatesas shown in FIG. 15 after co-crystallization with4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1), or the structure defined by the structure coordinatesas shown in FIG. 16 after co-crystallization with[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG).

Considering the above, the skilled person will readily understand thatsaid polypeptide fragment (variant) has a crystal structure which isonly defined by the structure coordinates of its amino acids andoptionally of its bound divalent cations comprised in FIG. 1, 2 to 5, 15or 16 and that it is not defined by the structure coordinates of therespective compound used for co-crystallization which are also comprisedin FIG. 2 to 5, 15 or 16, i.e. the compound EMBL-R05-3 has the residuedescriptor ci3 in FIGS. 2 and 3, the compound EMBL-R05-2 has the residuedescriptor cit in FIG. 4, the compound rUMP has the residue descriptor Uin FIG. 5, the compound EMBL-R05-1 has the residue descriptor ci1 inFIG. 15 and the compound EGCG has the residue descriptor tte in FIG. 16.

It is also preferred that said polypeptide fragment (variant) having thestructure defined by the structure coordinates as shown in FIG. 1 has acrystalline form with space group C2 and unit cell dimensions of a=26.36nm±0.5 nm, b=6.62 nm±0.3 nm, c=6.63 nm±0.3 nm, α=90 deg, β=96±2 deg,γ=90 deg, having the structure defined by the structure coordinates asshown in FIGS. 2 to 5 has a crystalline form with space group P2₁2₁2₁and unit cell dimensions of a=5.46±0.3 nm, b=12.25±0.4 nm, c=13.0±0.3nm, α=90 deg, β=90 deg, γ=90 deg, having the structure defined by thestructure coordinates as shown in FIG. 15 has a crystalline form withspace group P 6₂22 and unit cell dimensions of a=7.50 nm±0.3 nm, b=7.50nm±0.3 nm, c=12.00 nm±0.5 nm, α=90 deg, β=90 deg, γ=120 deg, or havingthe structure defined by the structure coordinates as shown in FIG. 16has a crystalline form with space group P6₄22 and unit cell dimensionsof a=9.99 nm±0.5 nm, b=9.99 nm±0.5 nm, c=8.27 nm±0.3 nm, α=90 deg, β=90deg, γ=120 deg.

Preferably, the crystal of the polypeptide fragment (variant) diffractsX-rays to a resolution of 2.8 Å or higher, preferably 2.6 Å or higher,more preferably 2.5 Å or higher, even more preferably 2.4 Å or higher,most preferably 2.1 Å or higher or 1.9 Å or higher. For example, thecrystal of the polypeptide fragment (variant) diffracts X-rays to aresolution of 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 Å orhigher.

It is another aspect of the present invention to provide an isolatedpolynucleotide coding for the above-mentioned (isolated) PA polypeptidefragments and variants thereof according to the present invention.

In a preferred embodiment of the present invention, the isolatedpolynucleotide codes for the PA polypeptide fragment which comprises SEQID NO:13 (the amino acids 1 to 198 of the amino acid sequence set forthin SEQ ID NO: 2).

In another preferred embodiment of the present invention, the isolatedpolynucleotide codes for the PA polypeptide fragment variant whichcomprises SEQ ID NO:14 (the amino acids 1 to 198 of the amino acidsequence set forth in SEQ ID NO: 2 with amino acids 52 to 64 replaced bythe amino acid glycine).

In a further preferred embodiment of the present invention, the isolatedpolynucleotide codes for the PA polypeptide fragment variant whichcomprises SEQ ID NO:15 (the amino acids 1 to 198 of the amino acidsequence set forth in SEQ ID NO: 2 with amino acids 52 to 72 replaced bythe amino acid glycine).

In another further preferred embodiment of the present invention, theisolated polynucleotide codes for the PA polypeptide fragment variantwhich comprises SEQ ID NO:16 (the amino acids 1 to 198 of the amino acidsequence set forth in SEQ ID NO: 2 with amino acids 52 to 69 replaced bythe amino acid glycine).

The molecular biology methods applied for obtaining such isolatednucleotide fragments are generally known to the person skilled in theart (for standard molecular biology methods see Sambrook et al., Eds.,“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), which is incorporated herein byreference). For example, RNA can be isolated from Influenza A 2009pandemic H1N1 virus infected cells and cDNA generated applying reversetranscription polymerase chain reaction (RT-PCR) using either randomprimers (e.g., random hexamers of decamers) or primers specific for thegeneration of the fragments of interest. The fragments of interest canthen be amplified by standard PCR using fragment specific primers.

The isolated polynucleotides coding for the Influenza A 2009 pandemicH1N1 virus RNA-dependent RNA polymerase PA subunit fragments are derivedfrom SEQ ID NO: 1. In this context, “derived” refers to the fact thatSEQ ID NO: 1 encodes the full-length PA polypeptide and, thus,polynucleotides coding for preferred PA polypeptide fragments maycomprise deletions at the 3′- and/or 5′-ends of the polynucleotides asrequired by the respectively encoded PA polypeptide fragments.

In another aspect, the present invention relates to a recombinant vectorcomprising the isolated polynucleotide according to the presentinvention. The person skilled in the art is well aware of techniquesused for the incorporation of polynucleotide sequences of interest intovectors (also see Sambrook et al., 1989, supra). Such vectors includeany vectors known to the skilled person including plasmid vectors,cosmid vectors, phage vectors such as lambda phage, viral vectors suchas adenoviral or baculoviral vectors, or artificial chromosome vectorssuch as bacterial artificial chromosomes (BAC), yeast artificialchromosomes (YAC), or P1 artificial chromosomes (PAC). Said vectors maybe expression vectors suitable for prokaryotic or eukaryotic expression.Said plasmids may include an origin of replication (ori), a multiplecloning site, and regulatory sequences such as promoter (constitutive orinducible), transcription initiation site, ribosomal binding site,transcription termination site, polyadenylation signal, and selectionmarker such as antibiotic resistance or auxotrophic marker based oncomplementation of a mutation or deletion. In one embodiment thepolynucleotide sequence of interest is operably linked to the regulatorysequences.

In another embodiment, said vector includes nucleotide sequences codingfor epitope-, peptide-, or protein-tags that facilitate purification ofpolypeptide fragments of interest. Such epitope-, peptide-, orprotein-tags include, but are not limited to, hemagglutinin-(HA-),FLAG-, myc-tag, poly-His-tag, glutathione-S-transferase-(GST-),maltose-binding-protein-(MBP-), NusA-, and thioredoxin-tag, orfluorescent protein-tags such as (enhanced) green fluorescent protein((E)GFP), (enhanced) yellow fluorescent protein ((E)YFP), redfluorescent protein (RFP) derived from Discosoma species (DsRed) ormonomeric (mRFP), cyan fluorescence protein (CFP), and the like. In apreferred embodiment, the epitope-, peptide-, or protein-tags can becleaved off the polypeptide fragment of interest, for example, using aprotease such as thrombin, Factor Xa, PreScission, TEV protease, and thelike. Preferably, the tag can be cleaved of with a TEV protease. Therecognition sites for such proteases are well known to the personskilled in the art. For example, the seven amino acid consensus sequenceof the TEV protease recognition site is Glu-X-X-Tyr-X-Gln-Gly/Ser,wherein X may be any amino acid and is in the context of the presentinvention preferably Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 12). Inanother embodiment, the vector includes functional sequences that leadto secretion of the polypeptide fragment of interest into the culturemedium of the recombinant host cells or into the periplasmic space ofbacteria. The signal sequence fragment usually encodes a signal peptidecomprised of hydrophobic amino acids which direct the secretion of theprotein from the cell. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria). Preferably there are processing sites, which can be cleavedeither in vivo or in vitro encoded between the signal peptide fragmentand the foreign gene.

In another aspect, the present invention provides a recombinant hostcell comprising the isolated polynucleotide according to the presentinvention or the recombinant vector according to the present invention.The recombinant host cells may be prokaryotic cells such as archea andbacterial cells or eukaryotic cells such as yeast, plant, insect, ormammalian cells. In a preferred embodiment the host cell is a bacterialcell such as an E. coli cell. The person skilled in the art is wellaware of methods for introducing said isolated polynucleotide or saidrecombinant vector into said host cell. For example, bacterial cells canbe readily transformed using, for example, chemical transformation,e.g., the calcium chloride method, or electroporation. Yeast cells maybe transformed, for example, using the lithium acetate transformationmethod or electroporation. Other eukaryotic cells can be transfected,for example, using commercially available liposome-based transfectionkits such as Lipofectamine™ (Invitrogen), commercially availablelipid-based transfection kits such as Fugene (Roche Diagnostics),polyethylene glycol-based transfection, calcium phosphate precipitation,gene gun (biolistic), electroporation, or viral infection. In apreferred embodiment of the invention, the recombinant host cellexpresses the polynucleotide fragment of interest. In an even morepreferred embodiment, said expression leads to soluble polypeptidefragments of the invention. These polypeptide fragments may be purifiedusing protein purification methods well known to the person skilled inthe art, optionally taking advantage of the above-mentioned epitope-,peptide-, or protein-tags.

In another aspect, the present invention relates to a method foridentifying compounds, which modulate the endonuclease activity of thePA subunit of a RNA-dependent RNA polymerase from Influenza A 2009pandemic H1N1 virus or a variant thereof comprising the steps of:

-   (a) constructing a computer model of the active site defined by (i)    the structure coordinates of the polypeptide fragment according to    the present invention as shown in FIG. 1, (ii) the structure    coordinates of the polypeptide fragment according to the present    invention as shown in FIG. 2, (iii) the structure coordinates of the    polypeptide fragment according to the present invention as shown in    FIG. 3, (iv) the structure coordinates of the polypeptide fragment    according to the present invention as shown in FIG. 4, (v) the    structure coordinates of the polypeptide fragment according to the    present invention as shown in FIG. 5, (vi) the structure coordinates    of the polypeptide fragment (variant) according to the present    invention as shown in FIG. 15, and/or (vii) the structure    coordinates of the polypeptide fragment (variant) according to the    present invention as shown in FIG. 16,-   (b) selecting a potential modulating compound by a method selected    from the group consisting of:

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound;

-   (c) employing computational means to perform a fitting program    operation between computer models of the said compound and the said    active site in order to provide an energy-minimized configuration of    the said compound in the active site; and-   (d) evaluating the results of said fitting operation to quantify the    association between the said compound and the active site model,    whereby evaluating the ability of said compound to associate with    the said active site.

In a preferred embodiment, a computer model of the active site definedby (i) the structure coordinates of the polypeptide fragment accordingto the present invention (i.e. amino acid structure coordinates) asshown in FIG. 1 and the structure coordinates of the divalent cations asshown in FIG. 1, (ii) the structure coordinates of the polypeptidefragment according to the present invention (i e amino acid structurecoordinates) as shown in FIG. 2 and the structure coordinates of thedivalent cations as shown in FIG. 2, (iii) the structure coordinates ofthe polypeptide fragment according to the present invention as shown inFIG. 3 and the structure coordinates of the divalent cations as shown inFIG. 3, (iv) the structure coordinates of the polypeptide fragmentaccording to the present invention as shown in FIG. 4 and the structurecoordinates of the divalent cations as shown in FIG. 4, (v) thestructure coordinates of the polypeptide fragment according to thepresent invention as shown in FIG. 5 and the structure coordinates ofthe divalent cations as shown in FIG. 5, (vi) the structure coordinatesof the polypeptide fragment (variant) according to the present invention(i e amino acid structure coordinates) as shown in FIG. 15 and thestructure coordinates of the divalent cations as shown in FIG. 15,and/or (vii) the structure coordinates of the polypeptide fragment(variant) according to the present invention (i e amino acid structurecoordinates) as shown in FIG. 16 and the structure coordinates of thedivalent cations as shown in FIG. 16 is constructed in step a) of themethod of the present invention.

Preferably, the modulating compound associates with, preferably bindsto, the endonucleolytically active site within the PA subunit of aRNA-dependent RNA polymerase from Influenza A 2009 pandemic H1N1 virusor variant thereof. The modulating compound may increase or decrease,preferably decrease said endonucleolytic activity.

In a preferred embodiment of this aspect of the present invention, thecompound that modulates the endonuclease activity of the PA subunit of aRNA-dependent RNA polymerase from Influenza A 2009 pandemic H1N1 virusor variant thereof decreases said activity, more preferably saidcompound inhibits said activity. Preferably, the compound decreases theendonucleolytic activity of the PA subunit of a RNA-dependent RNApolymerase from Influenza A 2009 pandemic H1N1 virus or variant thereofby 50%, more preferably by 60%, even more preferably by 70%, even morepreferably by 80%, even more preferably by 90%, and most preferably by100% compared to the endonucleolytic activity of said PA subunit or avariant thereof without said compound but with otherwise the samereaction conditions, i.e., buffer conditions, reaction time andtemperature. It is particularly preferred that the compound specificallydecreases or inhibits the endonucleolytic activity of the PA subunit ofa RNA-dependent RNA polymerase from Influenza A 2009 pandemic H1N1 virusor variant thereof but does not decrease or inhibit the endonucleolyticactivity of other endonucleases, in particular of mammalianendonucleases, to the same extent, preferably not at all.

The present invention permits the use of molecular design techniques toidentify, select, or design compounds that potentially modulate theendonucleolytic activity of the PA subunit of a RNA-dependent RNApolymerase from Influenza A 2009 pandemic H1N1 virus or variant thereof,based on the structure coordinates of the (native) endonucleolyticallyactive site according to FIG. 1. For the first time, the presentinvention further permits the optimized identification, selection anddesign of compounds that potentially modulate the endonucleolyticactivity of the PA subunit of a RNA-dependent RNA polymerase fromInfluenza A 2009 pandemic H1N1 virus or variant thereof, based on thestructure coordinates of the endonucleolytically active site accordingto FIG. 2 to 5, 15 or 16. Said structure coordinates have been achievedfrom polypeptide fragments (polypeptide fragment variants) according tothe present invention which have been co-crystallized with a modulatingcompound, preferably with4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3) (see FIGS. 2 and 3), with4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2) (see FIG. 4), with ribo-Uridine monophosphate (rUMP)(see FIG. 5), with4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1) (see FIG. 15), or with[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG) (see FIG. 16) (see also Tables 1 or 2). The present inventorshave namely surprisingly found that the structure coordinates of thepolypeptide fragment (variant) of the present invention without boundcompound (i.e. native polypeptide fragment (variant)) (see, for example,FIG. 1) differ from the structure coordinates of the polypeptidefragment (variant) of the present invention with bound compound (see,for example, FIG. 2 to 5, 15 or 16) as said polypeptide fragment(variant) changes its conformation after compound binding. Thus, thisnew three-dimensional knowledge allows the optimized design ofmodifications to existing inhibitors in order to improve their potencyor the design and optimization of novel inhibitors that effectivelyblock endonuclease activity.

Such predictive models are valuable in light of the higher costsassociated with the preparation and testing of the many diversecompounds that may possibly modulate the endonucleolytic activity. Inorder to use the structure coordinates generated for the Influenza A2009 pandemic H1N1 PA polypeptide fragment (variant) it is necessary toconvert the structure coordinates into a three-dimensional shape. Thisis achieved through the use of commercially available software that iscapable of generating three-dimensional graphical representations ofmolecules or portions thereof from a set of structure coordinates. Anexample for such a computer program is MODELER (Sali and Blundell, 1993,J. Mol. Biol. 234:779-815 as implemented in the Insight II Homologysoftware package (Insight II (97.0), Molecular Simulations Incorporated,San Diego, Calif.)).

One skilled in the art may use several methods to screen chemicalentities or fragments for their ability to modulate the endonucleolyticactivity of the Influenza A 2009 pandemic H1N1 PA subunit or Influenza A2009 pandemic H1N1 PA subunit PA polypeptide variants. This process maybegin by a visual inspection of, for example, a three-dimensionalcomputer model of the endonucleolytically active site of PA based on thestructural coordinates according to FIG. 1 to 5, 15 or 16. Selectedfragments or chemical compounds may then be positioned in a variety oforientations or docked within the active site. Docking may beaccomplished using software such as Cerius, Quanta, and Sybyl (TriposAssociates, St. Louis, Mo.), followed by energy minimization andmolecular dynamics with standard molecular dynamics force fields such asOPLS-AA, CHARMM, and AMBER. Additional specialized computer programsthat may assist the person skilled in the art in the process ofselecting suitable compounds or fragments include, for example, (i)AUTODOCK (Goodsell et al., 1990, Proteins: Struct., Funct., Genet. 8:195-202; AUTODOCK is available from The Scripps Research Institute, LaJolla, Calif.) and (ii) DOCK (Kuntz et al., 1982, J. Mol. Biol.161:269-288; DOCK is available from the University of California, SanFrancisco, Calif.).

Once suitable compounds or fragments have been selected, they can bedesigned or assembled into a single compound or complex. This manualmodel building is performed using software such as Quanta or Sybyl.Useful programs aiding the skilled person in connecting individualcompounds or fragments include, for example, (i) CAVEAT (Bartlett etal., 1989, in Molecular Recognition in Chemical and Biological Problems,Special Publication, Royal Chem. Soc. 78:182-196; Lauri and Bartlett,1994, J. Comp. Aid. Mol. Des. 8:51-66; CAVEAT is available from theUniversity of California, Berkley, Calif.), (ii) 3D Database systemssuch as ISIS (MDL Information Systems, San Leandro, Calif.; reviewed inMartin, 1992, J. Med. Chem. 35:2145-2154), and (iii) HOOK (Eisen et al.,1994, Proteins: Struct., Funct., Genet. 19:199-221; HOOK is availablefrom Molecular Simulations Incorporated, San Diego, Calif.).

Another approach enabled by this invention, is the computationalscreening of small molecule databases for compounds that can bind inwhole or part to the endonucleolytically active site of the Influenza A2009 pandemic H1N1 PA subunit or active sites of Influenza A 2009pandemic H1N1 PA polypeptide variants. In this screening, the quality offit of such compounds to the active site may be judged either by shapecomplementarity or by estimated interaction energy (Meng et al., 1992,J. Comp. Chem. 13:505-524).

Alternatively, a potential modulator for the endonucleolytic activity ofthe Influenza A 2009 pandemic H1N1 PA subunit or Influenza A 2009pandemic H1N1 polypeptide variant thereof, preferably an inhibitor ofthe endonucleolytic activity, may be designed de novo on the basis ofthe 3D structure of the PA polypeptide fragment according to FIGS. 1 to5. There are various de novo ligand design methods available to theperson skilled in the art. Such methods include (i) LUDI (Bohm, 1992, J.Comp. Aid. Mol. Des. 6:61-78; LUDI is available from MolecularSimulations Incorporated, San Diego, Calif.), (ii) LEGEND (Nishibata andItai, Tetrahedron 47:8985-8990; LEGEND is available from MolecularSimulations Incorporated, San Diego, Calif.), (iii) LeapFrog (availablefrom Tripos Associates, St. Louis, Mo.), (iv) SPROUT (Gillet et al.,1993, J. Comp. Aid. Mol. Des. 7:127-153; SPROUT is available from theUniversity of Leeds, UK), (v) GROUPBUILD (Rotstein and Murcko, 1993, J.Med. Chem. 36:1700-1710), and (vi) GROW (Moon and Howe, 1991, Proteins11:314-328).

In addition, several molecular modeling techniques (hereby incorporatedby reference) that may support the person skilled in the art in de novodesign and modeling of potential modulators and/or inhibitors of theendonucleolytically active site, preferably binding partners of theendonucleolytically active site, have been described and include, forexample, Cohen et al., 1990, J. Med. Chem. 33:883-894; Navia and Murcko,1992, Curr. Opin. Struct. Biol. 2:202-210; Balbes et al., 1994, Reviewsin Computational Chemistry, Vol. 5, Lipkowitz and Boyd, Eds., VCH, NewYork, pp. 37-380; Guida, 1994, Curr. Opin. Struct. Biol. 4:777-781.

A molecule designed or selected as binding to the endonucleolyticallyactive site of the Influenza A 2009 pandemic H1N1 PA subunit or variantsthereof may be further computationally optimized so that in its boundstate it preferably lacks repulsive electrostatic interaction with thetarget region. Such non-complementary (e.g., electrostatic) interactionsinclude repulsive charge-charge, dipole-dipole and charge-dipoleinteractions. Specifically, the sum of all electrostatic interactionsbetween the binding compound and the binding pocket in a bound state,preferably make a neutral or favorable contribution to the enthalpy ofbinding. Specific computer programs that can evaluate a compounddeformation energy and electrostatic interaction are available in theart. Examples of suitable programs include (i) Gaussian 92, revision C(Frisch, Gaussian, Incorporated, Pittsburgh, Pa.), (ii) AMBER, version4.0 (Kollman, University of California, San Francisco, Calif.), (iii)QUANTA/CHARMM (Molecular Simulations Incorporated, San Diego, Calif.),(iv) OPLS-AA (Jorgensen, 1998, Encyclopedia of Computational Chemistry,Schleyer, Ed., Wiley, New York, Vol. 3, pp. 1986-1989), and (v) InsightII/Discover (Biosysm Technologies Incorporated, San Diego, Calif.).These programs may be implemented, for instance, using a SiliconGraphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550.Other hardware systems and software packages are known to those skilledin the art.

Once a molecule of interest has been selected or designed, as describedabove, substitutions may then be made in some of its atoms or sidegroups in order to improve or modify its binding properties. Generally,initial substitutions are conservative, i.e., the replacement group willapproximate the same size, shape, hydrophobicity and charge as theoriginal group. It should, of course, be understood that componentsknown in the art to alter conformation should be avoided. Suchsubstituted chemical compounds may then be analyzed for efficiency offit to the endonucleolytically active site of the Influenza A 2009pandemic H1N1 PA subunit or variant thereof by the same computer methodsdescribed in detail above.

In one embodiment of the above-described method of the presentinvention, the endonucleolytically active site of the Influenza A 2009pandemic H1N1 PA subunit or variant thereof comprises amino acids Glu80,Glu119, Asp108, Ile120, and His41 of the PA subunit according to SEQ IDNO: 2 or amino acids corresponding thereto. In another embodiment, saidactive site comprises amino acids Glu80, Glu119, Asp108, Ile120, His41and Lys34 of the PA subunit according to SEQ ID NO: 2 or amino acidscorresponding thereto. In another embodiment, said active site comprisesamino acids Glu80, Glu119, Asp108, Ile120, His41 and Tyr24 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.In another embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41 and Arg84 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto. In anotherembodiment, said active site comprises amino acids Glu80, Glu119,Asp108, Ile120, His41 and Phe105 of the PA subunit according to SEQ IDNO: 2 or amino acids corresponding thereto. In another embodiment, saidactive site comprises amino acids Glu80, Glu119, Asp108, Ile120, His41,and Tyr130 of the PA subunit according to SEQ ID NO: 2 or amino acidscorresponding thereto. In another embodiment, said active site comprisesamino acids Glu80, Glu119, Asp108, Ile120, His41, and Ile38 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.In another embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, and Arg124 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto. In anotherembodiment, said active site comprises amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, and Tyr24 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto. In anotherembodiment, said active site comprises amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, and Arg84 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto. Inanother embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, and Phe105 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.In another embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, and Tyr130of the PA subunit according to SEQ ID NO: 2 or amino acids correspondingthereto. In another embodiment, said active site comprises amino acidsGlu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, and Ile38 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto. In another embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38 and Arg124 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto.

In yet another embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130,Ile38, and Glu26 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto, or amino acids Glu80, Glu119, Asp108,Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Arg124 andGlu26 of the PA subunit according to SEQ ID NO: 2 or amino acidscorresponding thereto. In yet another embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38, and Lys134 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto, or aminoacids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Arg124, and Lys134 of the PA subunit according to SEQ IDNO: 2 or amino acids corresponding thereto. In yet another embodiment,said active site comprises amino acids Glu80, Glu119, Asp108, Ile120,His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Leu106 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto,or amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24,Arg84, Phe105, Tyr130, Ile38, Arg124, and Leu106 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto. In yetanother embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130,Ile38, and Lys137 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto, or amino acids Glu80, Glu119, Asp108,Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Arg124, andLys137 of the PA subunit according to SEQ ID NO: 2 or amino acidscorresponding thereto. In yet another embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38, Gly26 and Lys134 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto, or aminoacids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Arg124, Gly26 and Lys134 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto. In yet anotherembodiment, said active site comprises amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38,Leu106, and Lys137 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto, or amino acids Glu80, Glu119, Asp108,Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Arg124,Leu106, and Lys137 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto. In yet another embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38, Glu26, and Lys137 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto, or aminoacids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Arg124, Glu26, and Lys137 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto. In yet anotherembodiment, said active site comprises amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38,Glu26, and Leu106 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto, or amino acids Glu80, Glu119, Asp108,Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Arg124,Glu26, and Leu106 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto. In yet another embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38, Lys134, and Leu106 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto,or amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24,Arg84, Phe105, Tyr130, Ile38, Arg124, Lys134, and Leu106 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.In yet another embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130,Ile38, Lys134, and Lys137 of the PA subunit according to SEQ ID NO: 2 oramino acids corresponding thereto, or amino acids Glu80, Glu119, Asp108,Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Arg124,Lys134, and Lys137 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto. In yet another embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38, Glu26, Lys134, and Leu106 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto,or amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24,Arg84, Phe105, Tyr130, Ile38, Arg124, Glu26, Lys134, and Leu106 of thePA subunit according to SEQ ID NO: 2 or amino acids correspondingthereto. In yet another embodiment, said active site comprises aminoacids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Glu26, Lys134, Leu106, and Lys137 of the PA subunitaccording to SEQ ID NO: 2 or amino acids corresponding thereto, or aminoacids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Arg124, Glu26, Lys134, Leu106, and Lys137 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.

In a further embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, and Glu26 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto. In a furtherembodiment, said active site comprises amino acids Glu80, Glu119,Asp108, Ile120, His41, and Lys134 of the PA subunit according to SEQ IDNO: 2 or amino acids corresponding thereto. In a further embodiment,said active site comprises amino acids Glu80, Glu119, Asp108, Ile120,His41, and Leu106 of the PA subunit according to SEQ ID NO: 2 or aminoacids corresponding thereto. In a further embodiment, said active sitecomprises amino acids Glu80, Glu119, Asp108, Ile120, His41, and Lys137of the PA subunit according to SEQ ID NO: 2 or amino acids correspondingthereto. In a further embodiment, said active site comprises amino acidsGlu80, Glu119, Asp108, Ile120, His41, Glu26, and Lys134 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.In a further embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Glu26, Lys134, and Leu106 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.In a further embodiment, said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Glu26, Lys134, Leu106, and Lys137 of thePA subunit according to SEQ ID NO: 2 or amino acids correspondingthereto.

In one embodiment of the above-described method of the invention, theendonucleolytically active site of the Influenza A 2009 pandemic H1N1 PAsubunit or variant thereof is defined by the structure coordinates ofthe PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120,and His41 according to FIGS. 1 to 5 or by the structure coordinates ofthe PA subunit amino acids Glu, Glu, Asp, Ile, and His corresponding toamino acids Glu80, Glu119, Asp108, Ile120, and His41 of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In another embodiment, saidactive site is defined by the structure coordinates of the PA subunitSEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41 and Lys34according to FIGS. 1 to 5 or by the structure coordinates of the PAsubunit amino acids Glu, Glu, Asp, Ile, His and Lys corresponding toamino acids Glu80, Glu119, Asp108, Ile120, and His41 and Lys34 of SEQ IDNO: 2, respectively, according to FIG. 15 or 16. In another embodiment,said active site is defined by the structure coordinates of the PAsubunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41and Tyr24 according to FIGS. 1 to 5 or by the structure coordinates ofthe PA subunit amino acids Glu, Glu, Asp, Ile, His, and Tyrcorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41 andTyr24 of SEQ ID NO: 2, respectively, according to FIG. 15 or 16. Inanother embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41 and Arg84 according to FIGS. 1 to 5 or by thestructure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile,His and Arg corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41 and Arg84 of SEQ ID NO: 2, respectively, according to FIG. 15 or16. In another embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41 and Phe105 according to FIGS. 1 to 5 or by thestructure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile,His and Phe corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41 and Phe105 of SEQ ID NO: 2, respectively, according to FIG. 15 or16. In another embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41 and Tyr130 according to FIGS. 1 to 5 or by thestructure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile,His and Tyr corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41 and Tyr130 of SEQ ID NO: 2, respectively, according to FIG. 15 or16. In another embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, and Ile38 according to FIGS. 1 to 5 or by thestructure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile,His and Ile corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41 and Ile38 of SEQ ID NO: 2, respectively, according to FIG. 15 or16. In another embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, and Arg124 according to FIGS. 1 to 5 or by thestructure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile,His, and Arg corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41, and Arg124 of SEQ ID NO: 2, respectively, according to FIG. 15 or16.

In another embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, and Tyr24 according to FIGS. 1 to 5 or bythe structure coordinates of the PA subunit amino acids Glu, Glu, Asp,Ile, His, Lys, and Tyr corresponding to amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, and Tyr24 of SEQ ID NO: 2, respectively,according to FIG. 15 or 16. In another embodiment, said active site isdefined by the structure coordinates of the PA subunit SEQ ID NO: 2amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, andArg84 according to FIGS. 1 to 5 or by the structure coordinates of thePA subunit amino acids Glu, Glu, Asp, Ile, His, Lys, Tyr, and Argcorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, and Arg84 of SEQ ID NO: 2, respectively, according to FIG.15 or 16. In another embodiment, said active site is defined by thestructure coordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, and Phe105 accordingto FIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, and Phe corresponding toamino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84,and Phe105 of SEQ ID NO: 2, respectively, according to FIG. 15 or 16. Inanother embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, and Tyr130 accordingto FIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, and Tyr correspondingto amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24,Arg84, Phe105, and Tyr130 of SEQ ID NO: 2, respectively, according toFIG. 15 or 16. In another embodiment, said active site is defined by thestructure coordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, andIle38 according to FIGS. 1 to 5 or by the structure coordinates of thePA subunit amino acids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr,and Ile corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, and Ile38 of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In another embodiment, saidactive site is defined by the structure coordinates of the PA subunitSEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34,Tyr24, Arg84, Phe105, Tyr130, Ile38, and Arg124 according to FIGS. 1 to5 or by the structure coordinates of the PA subunit amino acids Glu,Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, and Arg correspondingto amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24,Arg84, Phe105, Tyr130, Ile38, and Arg124 of SEQ ID NO: 2, respectively,according to FIG. 15 or 16.

In yet another embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, andGlu26 according to FIGS. 1 to 5 or by the structure coordinates of thePA subunit amino acids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr,Ile, and Glu corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Glu26 of SEQ IDNO: 2, respectively, according to FIG. 15 or 16. In yet anotherembodiment, said active site is defined by the structure coordinates ofthe PA subunit SEQ ID NO: 2amino acids Glu80, Glu119, Asp108, Ile120,His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Lys134 accordingto FIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, and Lyscorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Lys134 of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In yet another embodiment,said active site is defined by the structure coordinates of the PAsubunit SEQ ID NO: 2amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Leu106 according toFIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, and Leucorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Leu106, of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In yet another embodiment,said active site is defined by the structure coordinates of the PAsubunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Lys137 according toFIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, and Lyscorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Lys137 of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In yet another embodiment,said active site is defined by the structure coordinates of the PAsubunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Gly26 and Lys134 accordingto FIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, Glu, andLys corresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Glu26, and Lys134 of SEQ IDNO: 2, respectively, according to FIG. 15 or 16. In yet anotherembodiment, said active site is defined by the structure coordinates ofthe PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120,His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Leu106, and Lys137according to FIGS. 1 to 5 or by the structure coordinates of the PAsubunit amino acids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr,Ile, Leu, and Lys corresponding to amino acids Glu80, Glu119, Asp108,Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Leu106, andLys137 of SEQ ID NO: 2, respectively, according to FIG. 15 or 16. In yetanother embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38,Glu26, and Lys137 according to FIGS. 1 to 5 or by the structurecoordinates of the PA subunit amino acids Glu, Glu, Asp, Ile, His, Lys,Tyr, Arg, Phe, Tyr, Ile, Glu, and Lys corresponding to amino acidsGlu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Glu26, and Lys137 of SEQ ID NO: 2, respectively,according to FIG. 15 or 16. In yet another embodiment, said active siteis defined by the structure coordinates of the PA subunit SEQ ID NO: 2amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84,Phe105, Tyr130, Ile38, Glu26, and Leu106 according to FIGS. 1 to 5 or bythe structure coordinates of the PA subunit amino acids Glu, Glu, Asp,Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, Glu, and Leu, corresponding toamino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84,Phe105, Tyr130, Ile38, Glu26, and Leu106 of SEQ ID NO: 2, respectively,according to FIG. 15 or 16. In yet another embodiment, said active siteis defined by the structure coordinates of the PA subunit SEQ ID NO: 2amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84,Phe105, Tyr130, Ile38, Lys134, and Leu106 according to FIGS. 1 to 5 orby the structure coordinates of the PA subunit amino acids Glu, Glu,Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, Lys, and Leu correspondingto amino acids Glu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24,Arg84, Phe105, Tyr130, Ile38, Lys134, and Leu106 of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In yet another embodiment,said active site is defined by the structure coordinates of the PAsubunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Lys134, and Lys137 accordingto FIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, Lys, andLys corresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Lys134, and Lys137 of SEQ IDNO: 2, respectively, according to FIG. 15 or 16. In yet anotherembodiment, said active site is defined by the structure coordinates ofthe PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120,His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Glu26, Lys134, andLeu106 according to FIGS. 1 to 5 or by the structure coordinates of thePA subunit amino acids Glu, Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr,Ile, Glu, Lys, and Leu corresponding to amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38,Glu26, Lys134, and Leu106 of SEQ ID NO: 2, respectively, according toFIG. 15 or 16. In yet another embodiment, said active site is defined bythe structure coordinates of the PA subunit SEQ ID NO: 2 amino acidsGlu80, Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Glu26, Lys134, Leu106, and Lys137 according to FIGS. 1 to5 or by the structure coordinates of the PA subunit amino acids Glu,Glu, Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, Glu, Lys, Leu, and Lyscorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Glu26, Lys134, Leu106, andLys137 of SEQ ID NO: 2, respectively, according to FIG. 15 or 16. In yetanother embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38,Arg124, Glu26, Lys134, Leu106, and Lys137 according to FIGS. 1 to 5 orby the structure coordinates of the PA subunit amino acids Glu, Glu,Asp, Ile, His, Lys, Tyr, Arg, Phe, Tyr, Ile, Arg, Glu, Lys, Leu, and Lyscorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, Arg124, Glu26, Lys134,Leu106, and Lys137 of SEQ ID NO: 2, respectively, according to FIG. 15or 16.

In a further embodiment, said active site is defined by the structurecoordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119,Asp108, Ile120, His41, and Glu26 according to FIGS. 1 to 5 or by thestructure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile,His, and Glu corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41, and Glu26 of SEQ ID NO: 2, respectively, according to FIG. 15 or16. In a further embodiment, said active site is defined by thestructure coordinates of the PA subunit SEQ ID NO: 2 amino acids Glu80,Glu119, Asp108, Ile120, His41, and Lys134 according to FIGS. 1 to 5 orby the structure coordinates of the PA subunit amino acids Glu, Glu,Asp, Ile, His, and Lys corresponding to amino acids Glu80, Glu119,Asp108, Ile120, His41, and Lys134 of SEQ ID NO: 2, respectively,according to FIG. 15 or 16. In a further embodiment, said active site isdefined by the structure coordinates of the PA subunit SEQ ID NO: 2amino acids Glu80, Glu119, Asp108, Ile120, His41, and Leu106 accordingto FIGS. 1 to 5 or by the structure coordinates of the PA subunit aminoacids Glu, Glu, Asp, Ile, His, and Leu corresponding to amino acidsGlu80, Glu119, Asp108, Ile120, His41, and Leu106 of SEQ ID NO: 2,respectively, according to FIG. 15 or 16. In a further embodiment, saidactive site is defined by the structure coordinates of the PA subunitSEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, His41, andLys137 according to FIGS. 1 to 5 or by the structure coordinates of thePA subunit amino acids Glu, Glu, Asp, Ile, His, and Lys corresponding toamino acids Glu80, Glu119, Asp108, Ile120, His41, and Lys137 of SEQ IDNO: 2, respectively, according to FIG. 15 or 16. In a furtherembodiment, said active site is defined by the structure coordinates ofthe PA subunit SEQ ID NO: 2 amino acids Glu80, Glu119, Asp108, Ile120,His41, Glu26, and Lys134 according to FIGS. 1 to 5 or by the structurecoordinates of the PA subunit amino acids Glu, Glu, Asp, Ile, His, Glu,and Lys corresponding to amino acids Glu80, Glu119, Asp108, Ile120,His41, Glu26, and Lys134 of SEQ ID NO: 2, respectively, according toFIG. 15 or 16. In a further embodiment, said active site is defined bythe structure coordinates of the PA subunit SEQ ID NO: 2 amino acidsGlu80, Glu119, Asp108, Ile120, His41, Glu26, Lys134, and Leu106according to FIGS. 1 to 5 or by the structure coordinates of the PAsubunit amino acids Glu, Glu, Asp, Ile, His, Glu, Lys, and Leucorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Glu26, Lys134, and Leu106 of SEQ ID NO: 2, respectively, according toFIG. 15 or 16. In a further embodiment, said active site is defined bythe structure coordinates of the PA subunit SEQ ID NO: 2 amino acidsGlu80, Glu119, Asp108, Ile120, His41, Glu26, Lys134, Leu106, and Lys137according to FIGS. 1 to 5 or by the structure coordinates of the PAsubunit amino acids Glu, Glu, Asp, Ile, His, Glu, Lys, Leu, and Lyscorresponding to amino acids Glu80, Glu119, Asp108, Ile120, His41,Glu26, Lys134, Leu106, and Lys137 of SEQ ID NO: 2, respectively,according to FIG. 15 or 16.

The inventors of the present invention have surprisingly found that theamino acids His 41, Glu80, Asp108, Glu119, and Ile120 according to SEQID NO: 2 within the active site of Influenza A 2009 pandemic H1N1 PAsubunit are required for divalent cation binding (see for example FIGS.1 and 6), while the amino acids Tyr24, Lys34, Ile38, Arg84, Phe105, andTyr130 according to SEQ ID NO: 2 within the active site of Influenza A2009 pandemic H1N1 PA subunit are required for compound (i.e. inhibitor)binding in the structures shown in FIGS. 2 to 5 (see also FIGS. 7 to 10and Tables 1 and 2) and are those amino acids which conformation changesfrom the unligated structure (see FIGS. 1 and 6). In addition, theinventors of the present invention determined that the amino acidresidues Leu106, Lys134, Lys137 and Glue26 according to SEQ ID NO: 2within the active site of Influenza A 2009 pandemic H1N1 PA subunit arealso required to make contacts with the tested compounds (see Tables 1and 2, FIGS. 2 to 5 and FIGS. 7 to 10). See also FIGS. 15 to 19. Inaddition, the inventors of the present invention have found that theamino acid Arg124 within the active site of Influenza A 2009 pandemicH1N1 PA subunit is required for compound (i e inhibitor) interaction inthe structures shown in FIGS. 16 and 18. This three-dimensionalknowledge of the compound/ligand interacting residues and the plasticityof the active site is important for the optimised design ofmodifications to existing inhibitors in order to improve their potency.In addition, the design, identification and selection of new anti viralcompounds that interact with these amino acids is, thus, highlypreferable.

If computer modeling according to the methods described hereinaboveindicates binding of a compound to the active site of the Influenza A2009 pandemic H1N1 PA subunit or a variant thereof, said compound may besynthesized and optionally said compound or a pharmaceuticallyacceptable salt thereof may be formulated with one or morepharmaceutically acceptable excipient(s) and/or carrier(s). Thus, theabove-described method may comprise the further step of

-   (e) synthesizing said compound and optionally formulating said    compound or a pharmaceutically acceptable salt thereof with one or    more pharmaceutically acceptable excipient(s) and/or carrier(s).

Optionally, the ability of said compound or of a pharmaceuticallyacceptable salt thereof or of a formulation thereof to modulate,preferably decrease, more preferably inhibit, the endonucleolyticactivity of the Influenza A 2009 pandemic H1N1 PA subunit or variantthereof may be tested in vitro or in vivo comprising the further step of

-   (f) contacting said compound with the PA polypeptide fragment or    variant thereof according to the present invention or the    recombinant host cell according to the present invention and    determine the ability of said compound to (i) bind to the active    site and/or (ii) modulate, preferably decrease, more preferably    inhibit, the endonucleolytic activity of the Influenza A 2009    pandemic H1N1 PA subunit polypeptide fragment or variant thereof.    The quality of fit of such compounds to the active site may be    judged either by shape complementarity or by estimated interaction    energy (Meng et al., 1992, J. Comp. Chem. 13:505-524). Methods for    synthesizing said compounds are well known to the person skilled in    the art or such compounds may be commercially available.

It is another aspect of the present invention to provide a compoundwhich is able to modulate, preferably to decrease, more preferably toinhibit, the endonuclease activity of the Influenza A 2009 pandemic H1N1PA subunit or variant thereof according to the present invention. It ispreferred that the compound is able to modulate, preferably to decrease,more preferably to inhibit, the endonuclease activity of the PA subunitor variant thereof according to the present invention by associatingwith, preferably by binding to, the endonucleolytically active site ofsaid PA subunit or variant thereof.

It is also an aspect of the present invention to provide a compoundwhich is able to modulate, preferably to decrease, more preferably toinhibit, the endonuclease activity of the Influenza A 2009 pandemic H1N1PA subunit polypeptide fragment or variant thereof according to thepresent invention. It is preferred that the compound is able tomodulate, preferably to decrease, more preferably to inhibit, theendonuclease activity of the PA subunit polypeptide fragment or variantthereof according to the present invention by associating with,preferably by binding to, the endonucleolytically active site of said PAsubunit polypeptide fragment or variant thereof.

It is further an aspect of the present invention to provide a compoundwhich is able to modulate, preferably to decrease, more preferably toinhibit, the endonuclease activity of the PA subunit of other viruses,preferably of viruses of the Orthomyxoviridae family, Bunyaviridaefamily and/or Arenviridae family. It is preferred that the compound isable to modulate, preferably to decrease, more preferably to inhibit,the endonuclease activity of the PA subunit of other viruses, preferablyof viruses of the Orthomyxoviridae family, Bunyaviridae family and/orArenviridae family, by associating with, preferably by binding to, theendonucleolytically active site of said PA subunit.

Preferably, the endonucleolytically active site of the above-mentionedPA subunits or variants thereof, or PA subunit polypeptide fragments orvariants thereof has the structure as defined above.

Thus, it is preferred that said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, and His41 of the PA subunit according to SEQ IDNO: 2 or amino acids corresponding thereto.

It is more preferred that said active site comprises amino acids Glu80,Glu119, Asp108, Ile120, His41, Lys34, Tyr24, Arg84, Phe105, Tyr130, andIle38 of the PA subunit according to SEQ ID NO: 2 or amino acidscorresponding thereto, or comprises amino acids Glu80, Glu119, Asp108,Ile120, His41 Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Arg124 ofthe PA subunit according to SEQ ID NO: 2 or amino acids correspondingthereto.

It is also more preferred that said active site comprises amino acidsGlu80, Glu119, Asp108, Ile120, His41, Glu26, Lys134, Leu106, and Lys137of the PA subunit according to SEQ ID NO: 2 or amino acids correspondingthereto.

It is most preferred that said active side comprises amino acids Glu80,Glu119, Asp108, Ile120, His41 Lys34, Tyr24, Arg84, Phe105, Tyr130,Ile38, Glu26, Lys134, Leu106, and Lys137 of the PA subunit according toSEQ ID NO: 2 or amino acids corresponding thereto, or comprises aminoacids Glu80, Glu119, Asp108, Ile120, His41 Lys34, Tyr24, Arg84, Phe105,Tyr130, Ile38, Arg124, Glu26, Lys134, Leu106, and Lys137 of the PAsubunit according to SEQ ID NO: 2 or amino acids corresponding thereto.

Preferably, the endonucleolytically active site of the above-mentionedPA subunits or variants thereof, or PA subunit polypeptide fragments orvariants thereof is defined by the structure coordinates of the PAsubunit as mentioned above.

The ability of a compound to associate with, preferably to bind to, theendonucleolytically active site of the afore-mentioned PA subunit orvariant thereof, or PA subunit polypeptide fragment or variant thereof,and/or to modulate, preferably to decrease, more preferably to inhibitthe endonucleolytic activity of the PA subunit or variant thereof, or PAsubunit polypeptide fragment or variant thereof can easily be assessed.For example, the purified PA subunit or PA subunit polypeptide fragmentand a substrate thereof such as panhandle RNA or single stranded DNA arecontacted in presence or absence of varying amounts of the test compoundand incubated for a certain period of time, for example, for 5, 10, 15,20, 30, 40, 60, or 90 minutes. The reaction conditions are chosen suchthat the PA subunit or PA subunit polypeptide fragment isendonucleolytically active without the test compound. The substrate isthen analyzed for degradation/endonucleolytic cleavage, for example, bygel electrophoresis. Alternatively, such a test may comprise a labeledsubstrate molecule which provides a signal when the substrate moleculeis endonucleolytically cleaved but does not provide a signal if it isintact. For example, the substrate polynucleotide chain may be labeledwith fluorescent reporter molecule and a fluorescence quencher such thatthe fluorescent reporter is quenched as long as the substratepolynucleotide chain is intact. In case the substrate polynucleotidechain is cleaved, the fluorescent reporter and the quencher areseparated, thus, the fluorescent reporter emits a signal which may bedetected, for example, by an ELISA reader. Further suitable methods aredescribed below.

Preferably, said compound is identifiable by the above-described method.More preferably, said compound is identified by the above-describedmethod.

The compound identifiable by the above-described method may be able tomodulate the endonuclease activity of the Influenza A 2009 pandemic H1N1PA subunit or variant thereof. The compound identifiable by theabove-described method may be able to modulate, preferably to decrease,more preferably to inhibit, the endonuclease activity of the Influenza A2009 pandemic H1N1 PA subunit polypeptide fragment or variant thereofaccording to the present invention. It is also possible that thecompound identifiable by the above-mentioned method may be able tomodulate, preferably to decrease, most preferably to inhibit, theendonuclease activity of the PA subunit of other viruses, preferably ofviruses of the Orthomyxoviridae family, Bunyaviridae family and/orArenviridae family.

Compounds of the present invention can be any agents including, but notrestricted to, peptides, peptoids, polypeptides, proteins (includingantibodies), lipids, metals, nucleotides, nucleosides, nucleic acids,small organic or inorganic molecules, chemical compounds, elements,saccharides, isotopes, carbohydrates, imaging agents, lipoproteins,glycoproteins, enzymes, analytical probes, polyamines, and combinationsand derivatives thereof. The term “small molecules” refers to moleculesthat have a molecular weight between 50 and about 2,500 Daltons,preferably in the range of 200-800 Daltons. In addition, a test compoundaccording to the present invention may optionally comprise a detectablelabel. Such labels include, but are not limited to, enzymatic labels,radioisotope or radioactive compounds or elements, fluorescent compoundsor metals, chemiluminescent compounds and bioluminescent compounds.

In a preferred embodiment of the compound according to the presentinvention, the compound is not a 4-substituted 2-dioxobutanoic acid, a4-substituted 4-dioxobutanoic acid, a 4-substituted 2,4-dioxobutanoicacid, a 2,6-diketopiperazine or a derivative thereof, a substituted2,6-diketopiperazine or a derivative thereof, pyrazine-2,6-dione or asubstituted pyrazine-2,6-dione such as flutimide, an N-hydroxamic acid,or an N-hydroxymide.

In particular, the compound according to the present invention is not acompound according to Formula I:

In another preferred embodiment of the compound according to the presentinvention, the compound is not4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid,4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid,4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid, ribo-Uridine Monophosphate (rUMP), desoxy-thymidinmonophosphat(dTMP), or 2.4-dioxo-4-phenylbutanoic acid (DPBA).

In a further preferred embodiment of the compound according to thepresent invention, the compound is not a catechin, preferably not a(−)-epigallocatechin gallate (EGCG), such as[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate,a (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC),(−)-epicatechin (EC), or gallic acid (GA). In another preferredembodiment of the compound of the present invention, the compound is nota phenethylphenylphthalimide, a phenethylphenylphthalimide analogderived from thalidomide or raltegravir (also a diketobutanoic acidderivative). In a further aspect, the present invention provides a (anin vitro) method for identifying compounds, which modulate, preferablydecrease, more preferably inhibit, the endonuclease activity of the PAsubunit of a RNA-dependent RNA polymerase from Influenza A 2009 pandemicH1N1 virus or a variant thereof comprising the steps of:

-   (i) contacting said polypeptide fragment or variant thereof    according to the present invention or said recombinant host cell    according to the present invention with a test compound, and-   (ii) analyzing the ability of said test compound to modulate,    preferably to decrease, more preferably to inhibit, the endonuclease    activity of said PA subunit polypeptide fragment or variant thereof.

It is preferred that said compounds modulate, preferably decrease, morepreferably inhibit, the endonuclease activity of the PA subunit of aRNA-dependent RNA polymerase from Influenza A 2009 pandemic H1N1 virusor a variant thereof by associating with, preferably by binding to, theendonucleolytically active site of said PA subunit.

In another further aspect, the present invention provides a (an invitro) method for identifying compounds, which bind to theendonucleolytically active site, (and) preferably modulate, morepreferably decrease, most preferably inhibit, the endonuclease activityof the PA subunit of a RNA-dependent RNA polymerase from Influenza A2009 pandemic H1N1 virus or a variant thereof comprising the steps of:

-   (i) contacting said polypeptide fragment or variant thereof    according to the present invention or said recombinant host cell    according to the present invention with a test compound, and-   (ii) analyzing the ability of said test compound to bind to the    endonucleolytically site, (and) preferably to modulate, more    preferably to decrease, most preferably to inhibit, the endonuclease    activity of said PA subunit polypeptide fragment or variant thereof.

In one embodiment, the interaction between the Influenza A 2009 pandemicH1N1 PA polypeptide fragment or variant thereof and a test compound maybe analyzed in form of a pull down assay. For example, the PApolypeptide fragment or variant thereof according to the invention maybe purified and may be immobilized on beads. In one embodiment, the PApolypeptide fragment immobilized on beads may be contacted, for example,with (i) another purified protein, polypeptide fragment, or peptide,(ii) a mixture of proteins, polypeptide fragments, or peptides, or (iii)a cell or tissue extract, and binding of proteins, polypeptidefragments, or peptides may be verified by polyacrylamide gelelectrophoresis in combination with coomassie staining or Westernblotting. Unknown binding partners may be identified by massspectrometric analysis.

In another embodiment, the interaction between the Influenza A 2009pandemic H1N1 PA polypeptide fragment or variant thereof and a testcompound may be analyzed in form of an enzyme-linked immunosorbent assay(ELISA)-based experiment. In one embodiment, the PA polypeptide fragmentor variant thereof according to the invention may be immobilized on thesurface of an ELISA plate and contacted with the test compound. Bindingof the test compound may be verified, for example, for proteins,polypeptides, peptides, and epitope-tagged compounds by antibodiesspecific for the test compound or the epitope-tag. These antibodiesmight be directly coupled to an enzyme or detected with a secondaryantibody coupled to said enzyme that—in combination with the appropriatesubstrates—carries out chemiluminescent reactions (e.g., horseradishperoxidase) or colorimetric reactions (e.g., alkaline phosphatase). Inanother embodiment, binding of compounds that cannot be detected byantibodies might be verified by labels directly coupled to the testcompounds. Such labels may include enzymatic labels, radioisotope orradioactive compounds or elements, fluorescent compounds or metals,chemiluminescent compounds and bioluminescent compounds. In anotherembodiment, the test compounds might be immobilized on the ELISA plateand contacted with the PA polypeptide fragment or variants thereofaccording to the invention. Binding of said polypeptide may be verifiedby a PA polypeptide fragment specific antibody and chemiluminescence orcolorimetric reactions as described above.

In a further embodiment, purified Influenza A 2009 pandemic H1N1 PApolypeptide fragments or variants may be incubated with a peptide arrayand binding of the PA polypeptide fragments to specific peptide spotscorresponding to a specific peptide sequence may be analyzed, forexample, by PA polypeptide specific antibodies, antibodies that aredirected against an epitope-tag fused to the PA polypeptide fragment, orby a fluorescence signal emitted by a fluorescent tag coupled to the PApolypeptide fragment.

In another embodiment, the recombinant host cell according to thepresent invention is contacted with a test compound. This may beachieved by co-expression of test proteins or polypeptides andverification of interaction, for example, by fluorescence resonanceenergy transfer (FRET) or co-immunoprecipitation. In another embodiment,directly labeled test compounds may be added to the medium of therecombinant host cells. The potential of the test compound to penetratemembranes and bind to the PA polypeptide fragment may be, for example,verified by immunoprecipitation of said polypeptide and verification ofthe presence of the label.

In another embodiment, the ability of the test compound to modulate,preferably decrease, more preferably inhibit the endonucleolyticactivity of the Influenza A 2009 pandemic H1N1 PA subunit polypeptidefragment or variant thereof is assessed. For example, the purified PAsubunit polypeptide fragment and a substrate thereof such as panhandleRNA or single stranded DNA are contacted in presence or absence ofvarying amounts of the test compound and incubated for a certain periodof time, for example, for 5, 10, 15, 20, 30, 40, 60, or 90 minutes. Thereaction conditions are chosen such that the PA subunit polypeptide isendonucleolytically active without the test compound. The substrate isthen analyzed for degradation/endonucleolytic cleavage, for example, bygel electrophoresis. Alternatively, such a test may comprise a labeledsubstrate molecule which provides a signal when the substrate moleculeis endonucleolytically cleaved but does not provide a signal if it isintact. For example, the substrate polynucleotide chain may be labeledwith fluorescent reporter molecule and a fluorescence quencher such thatthe fluorescent reporter is quenched as long as the substratepolynucleotide chain is intact. In case the substrate polynucleotidechain is cleaved, the fluorescent reporter and the quencher areseparated, thus, the fluorescent reporter emits a signal which may bedetected, for example, by an ELISA reader. This experimental setting maybe applied in a multi-well plate format and is suitable for highthroughput screening of compounds regarding their ability to modulate,decrease, or inhibit the endonuclease activity of the Influenza A 2009pandemic H1N1 PA subunit polypeptide fragment or variants thereof.

Preferably, the ability of the test compound to inhibit the endonucleaseactivity of the Influenza A 2009 pandemic H1N1 PA subunit polypeptidefragment or variant thereof is analyzed in the above-described methods.

In a preferred embodiment, the above-described methods for identifyingcompounds which associate with, preferably bind to, theendonucleolytically active site, modulate, decrease, and/or inhibit theendonucleolytic activity of the Influenza A 2009 pandemic H1N1 PAsubunit polypeptide fragment or variant thereof are performed in ahigh-throughput setting. In a preferred embodiment, said methods arecarried out in a multi-well microtiter plate as described above using PApolypeptide fragments or variants thereof according to the presentinvention and labeled test compounds.

In a preferred embodiment, the test compounds are derived from librariesof synthetic or natural compounds. For instance, synthetic compoundlibraries are commercially available from Maybridge Chemical Co.(Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, Calif.),or Aldrich (Milwaukee, Wis.). A natural compound library is, forexample, available from TimTec LLC (Newark, Del.). Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts can be used. Additionally, test compounds can besynthetically produced using combinatorial chemistry either asindividual compounds or as mixtures.

In another embodiment, the inhibitory effect of the identified compoundon the Influenza A 2009 pandemic H1N1 virus life cycle may be tested inan in vivo setting. A cell line that is susceptible for Influenza virusinfection such as 293T human embryonic kidney cells, Madin-Darby caninekidney cells, or chicken embryo fibroblasts may be infected withInfluenza A 2009 pandemic H1N1 virus in presence or absence of theidentified compound. In a preferred embodiment, the identified compoundmay be added to the culture medium of the cells in variousconcentrations. Viral plaque formation may be used as read out for theinfectious capacity of the Influenza A 2009 pandemic H1N1 virus and maybe compared between cells that have been treated with the identifiedcompound and cells that have not been treated.

In a further embodiment of the invention, the test compound applied inany of the above described methods is a small molecule. In a preferredembodiment, said small molecule is derived from a library, e.g., a smallmolecule inhibitor library. In another embodiment, said test compound isa peptide or protein. In a preferred embodiment, said peptide or proteinis derived from a peptide or protein library.

In another embodiment of the above-described methods for computationalas well as in vitro identification of compounds that associate with,preferably bind to, the endonucleolytically active site, modulate,decrease, and/or inhibit the endonucleolytic activity of the Influenza A2009 pandemic H1N1 PA subunit polypeptide fragment or variant thereofaccording to the present invention, said methods further comprise thestep of formulating said compound or a pharmaceutically acceptable saltthereof with one or more pharmaceutically acceptable excipient(s) and/orcarrier(s).

As already mentioned above, it is an aspect of the present invention toprovide a compound which is able to modulate, preferably to decrease,more preferably to inhibit, the endonuclease activity of the Influenza A2009 pandemic H1N1 PA subunit or variant thereof according to thepresent invention. It is preferred that the compound is able tomodulate, preferably to decrease, more preferably to inhibit, theendonuclease activity of the PA subunit or variant thereof according tothe present invention by associating with, preferably by binding to, theendonucleolytically active site of said PA subunit or variant thereof.

It is also an aspect of the present invention to provide a compoundwhich is able to modulate, preferably to decrease, more preferably toinhibit, the endonuclease activity of the Influenza A 2009 pandemic H1N1PA subunit polypeptide fragment or variant thereof according to thepresent invention. It is preferred that the compound is able tomodulate, preferably to decrease, more preferably to inhibit, theendonuclease activity of the PA subunit polypeptide fragment or variantthereof according to the present invention by associating with,preferably by binding to, the endonucleolytically active site of said PAsubunit polypeptide fragment or variant thereof.

It is further an aspect of the present invention to provide a compoundwhich is able to modulate, preferably to decrease, more preferably toinhibit, the endonuclease activity of the PA subunit of other viruses,preferably of viruses of the Orthomyxoviridae family, Bunyaviridaefamily and/or Arenviridae family. It is preferred that the compound isable to modulate, preferably to decrease, more preferably to inhibit,the endonuclease activity of the PA subunit of other viruses, preferablyof viruses of the Orthomyxoviridae family, Bunyaviridae family and/orArenviridae family, by associating with, preferably by binding to, theendonucleolytically active site of said PA subunit.

The ability of a compound to associate with, preferably to bind to, theendonucleolytically active site of the afore-mentioned PA subunit orvariant thereof, or PA subunit polypeptide fragment or variant thereof,and/or to modulate, preferably to decrease, more preferably to inhibitthe endonucleolytic activity of the PA subunit or variant thereof, or PAsubunit polypeptide fragment or variant thereof can easily be assessed.For example, the purified PA subunit or PA subunit polypeptide fragmentand a substrate thereof such as panhandle RNA or single stranded DNA arecontacted in presence or absence of varying amounts of the test compoundand incubated for a certain period of time, for example, for 5, 10, 15,20, 30, 40, 60, or 90 minutes. The reaction conditions are chosen suchthat the PA subunit or PA subunit polypeptide fragment isendonucleolytically active without the test compound. The substrate isthen analyzed for degradation/endonucleolytic cleavage, for example, bygel electrophoresis. Alternatively, such a test may comprise a labeledsubstrate molecule which provides a signal when the substrate moleculeis endonucleolytically cleaved but does not provide a signal if it isintact. For example, the substrate polynucleotide chain may be labeledwith fluorescent reporter molecule and a fluorescence quencher such thatthe fluorescent reporter is quenched as long as the substratepolynucleotide chain is intact. In case the substrate polynucleotidechain is cleaved, the fluorescent reporter and the quencher areseparated, thus, the fluorescent reporter emits a signal which may bedetected, for example, by an ELISA reader. Further suitable methods aredescribed below.

Preferably, said compound is identifiable by the above-described (invitro) methods. More preferably, said compound is identified by theabove-described (in vitro) methods.

The compound identifiable by the above described (in vitro) methods maybe able to modulate the endonuclease activity of the Influenza A 2009pandemic H1N1 PA subunit or variant thereof. The compound identifiableby the above-described (in vitro) methods may be able to modulate,preferably to decrease, more preferably to inhibit, the endonucleaseactivity of the Influenza A 2009 pandemic H1N1 PA subunit polypeptidefragment or variant thereof according to the present invention.

It is also possible that the compound identifiable by theabove-described (in vitro) methods may be able to modulate, preferablyto decrease, most preferably to inhibit, the endonuclease activity ofthe PA subunit of other viruses, preferably of viruses of theOrthomyxoviridae family, Bunyaviridae family and/or Arenviridae family.

Compounds of the present invention can be any agents as described abovefor the in silico screening methods. In a preferred embodiment of thecompound according to the present invention, the compound is not a4-substituted 2-dioxobutanoic acid, a 4-substituted 4-dioxobutanoicacid, a 4-substituted 2,4-dioxobutanoic acid, 2,6-diketopiperazine or aderivative thereof, a substituted 2,6-diketopiperazine or a derivativethereof, a pyrazine-2,6-dione or a substituted pyrazine-2,6-dione suchas flutimide, an N-hydroxamic acid, or an N-hydroxymide.

In particular, the compound according to the present invention is not acompound according to Formula I:

In another preferred embodiment of the compound according to the presentinvention, the compound is not4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid,4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid,4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid, ribo-Uridine Monophosphate (rUMP), desoxy-thymidinmonophosphat(dTMP), or 2.4-dioxo-4-phenylbutanoic acid (DPBA).

In a further preferred embodiment of the compound according to thepresent invention, the compound is not a catechin, preferably not a(−)-epigallocatechin gallate (EGCG), such as[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate,a (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC),(−)-epicatechin (EC), or gallic acid (GA). In another preferredembodiment of the compound of the present invention, the compound is nota phenethylphenylphthalimide, a phenethylphenylphthalimide analogderived from thalidomide or raltegravir (also a diketobutanoic acidderivative).

It is an aspect of the present invention to provide a pharmaceuticalcomposition comprising the afore-mentioned compound of the presentinvention or a pharmaceutically acceptable salt thereof. It is also anaspect of the present invention to provide a pharmaceutical compositioncomprising the afore-mentioned compound of the present invention or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable excipient(s) and/or carrier(s).

As already mentioned above, the compound comprised in the pharmaceuticalcompositions of the present invention (i) is able to modulate,preferably to decrease, more preferably to inhibit, the endonucleaseactivity of the Influenza A 2009 pandemic H1N1 PA subunit or variantthereof according to the present invention, e.g. by binding to theendonucleolytically active site of said subunit or variant thereof, (ii)is able to modulate, preferably to decrease, more preferably to inhibit,the endonuclease activity of the Influenza A 2009 pandemic H1N1 PAsubunit polypeptide fragment or variant thereof according to the presentinvention, e.g. by binding to the endonucleolytically active site ofsaid polypeptide fragment or variant thereof, or (iii) is able tomodulate, preferably to decrease, more preferably to inhibit, theendonuclease activity of the PA subunit of other viruses, preferably ofviruses of the Orthomyxoviridae family, Bunyaviridae family and/orArenviridae family, e.g. by binding to the endonucleolytically activesite of said PA subunit. Preferably, the compound comprised in thepharmaceutical compositions of the present invention is identifiable bythe above-described methods. More preferably, the compound comprised inthe pharmaceutical compositions of the present invention is identifiedby the above-described methods.

Preferably, said pharmaceutical composition(s) is (are) producibleaccording to the afore-mentioned methods.

A compound according to the present invention can be administered alonebut, in human therapy, will generally be administered in admixture witha suitable pharmaceutical excipient, diluent, or carrier selected withregard to the intended route of administration and standardpharmaceutical practice (see hereinafter).

In the aspect of computational modeling or screening of a bindingpartner for the endonucleolytically active site, a modulator, and/orinhibitor of the endonucleolytic activity of the Influenza A 2009pandemic H1N1 PA subunit polypeptide fragment or variant thereofaccording to the present invention, it may be possible to introduce intothe molecule of interest, chemical moieties that may be beneficial for amolecule that is to be administered as a pharmaceutical. For example, itmay be possible to introduce into or omit from the molecule of interest,chemical moieties that may not directly affect binding of the moleculeto the target area but which contribute, for example, to the overallsolubility of the molecule in a pharmaceutically acceptable carrier, thebioavailability of the molecule and/or the toxicity of the molecule.Considerations and methods for optimizing the pharmacology of themolecules of interest can be found, for example, in “Goodman andGilman's The Pharmacological Basis of Therapeutics”, 8^(th) Edition,Goodman, Gilman, Rall, Nies, & Taylor, Eds., Pergamon Press (1985);Jorgensen & Duffy, 2000, Bioorg. Med. Chem. Lett 10:1155-1158.Furthermore, the computer program “Qik Prop” can be used to providerapid predictions for physically significant descriptions andpharmaceutically-relevant properties of an organic molecule of interest.A ‘Rule of Five’ probability scheme can be used to estimate oralabsorption of the newly synthesized compounds (Lipinski et al., 1997,Adv. Drug Deliv. Rev. 23:3-25). Programs suitable for pharmacophoreselection and design include (i) DISCO (Abbot Laboratories, Abbot Park,Ill.), (ii) Catalyst (Bio-CAD Corp., Mountain View, Calif.), and (iii)Chem DBS-3D (Chemical Design Ltd., Oxford, UK).

The pharmaceutical composition contemplated by the present invention maybe formulated in various ways well known to one of skill in the art. Forexample, the pharmaceutical composition of the present invention may bein solid form such as in the form of tablets, pills, capsules (includingsoft gel capsules), cachets, lozenges, ovules, powder, granules, orsuppositories, or in liquid form such as in the form of elixirs,solutions, emulsions, or suspensions.

Solid administration forms may contain excipients such asmicrocrystalline cellulose, lactose, sodium citrate, calcium carbonate,dibasic calcium phosphate, glycine, and starch (preferably corn, potato,or tapioca starch), disintegrants such as sodium starch glycolate,croscarmellose sodium, and certain complex silicates, and granulationbinders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose(HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia.Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate, and talc may be included. Solid compositions ofa similar type may also be employed as fillers in gelatin capsules.Preferred excipients in this regard include lactose, starch, acellulose, milk sugar, or high molecular weight polyethylene glycols.

For aqueous suspensions, solutions, elixirs, and emulsions suitable fororal administration the compound may be combined with various sweeteningor flavoring agents, coloring matter or dyes, with emulsifying and/orsuspending agents and with diluents such as water, ethanol, propyleneglycol, and glycerin, and combinations thereof.

The pharmaceutical composition of the present invention may containrelease rate modifiers including, for example, hydroxypropylmethylcellulose, methyl cellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer,ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax,paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulosephthalate, methacrylic acid copolymer, and mixtures thereof.

The pharmaceutical composition of the present invention may be in theform of fast dispersing or dissolving dosage formulations (FDDFs) andmay contain the following ingredients: aspartame, acesulfame potassium,citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethylacrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose,magnesium stearate, mannitol, methyl methacrylate, mint flavoring,polyethylene glycol, fumed silica, silicon dioxide, sodium starchglycolate, sodium stearyl fumarate, sorbitol, xylitol.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

The pharmaceutical composition of the present invention suitable forparenteral administration is best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary.

The pharmaceutical composition suitable for intranasal administrationand administration by inhalation is best delivered in the form of a drypowder inhaler or an aerosol spray from a pressurized container, pump,spray or nebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A™) or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA™), carbon dioxide, oranother suitable gas. The pressurized container, pump, spray ornebulizer may contain a solution or suspension of the active compound,e.g., using a mixture of ethanol and the propellant as the solvent,which may additionally contain a lubricant, e.g., sorbitan trioleate.

In another aspect, the present invention provides an antibody directedagainst the active site of the PA subunit of the Influenza A 2009pandemic H1N1 virus according to SEQ ID NO: 2 or a variant thereof,wherein said variant comprises the amino acid serine at an amino acidposition 186 according to SEQ ID NO: 2 or at an amino acid positioncorresponding thereto.

In a preferred embodiment, said antibody recognizes the endonucleasedomain by recognition of a polypeptide fragment selected from the groupof polypeptides defined by SEQ ID NO: 4 to 11, i.e., amino acids 20 to30 (SEQ ID NO: 4), 32 to 40 (SEQ ID NO: 5), 41 to 51 (SEQ ID NO: 6), 80to 90 (SEQ ID NO: 7), 100 to 110 (SEQ ID NO: 8), 115 to 125 (SEQ ID NO:9), 130 to 140 (SEQ ID NO: 10), and 176 to 186 (SEQ ID NO: 11) of theamino acid sequence as set forth in SEQ ID NO: 2.

In particular, said antibody specifically binds to an epitope comprisingone or more of above indicated amino acids, which define the activesite. In this context, the term epitope has its art recognized meaningand preferably refers to stretches of 4 to 20 amino acids, preferably 5to 18, 5 to 15, or 7 to 14 amino acids, e.g. stretches of 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.Accordingly, preferred epitopes have a length of 4 to 20, 5 to 18,preferably 5 to 15, or 7 to 14 amino acids, e.g. have a length of 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids,and/or comprise one or more of Lys34, Glu26, Ile38, Tyr24, His41, Glu80,Arg84, Leu106, Asp108, Glu119, Ile120, Arg124, Tyr130, Lys134, Phe105,and Lys137 of SEQ ID NO: 2 or one or more corresponding amino acid(s).Thus, in a preferred embodiment, said antibody recognizes a polypeptidefragment of a length between 5 and 15 amino acids of the amino acidsequence as set forth in SEQ ID NO: 2, wherein the polypeptide fragmentcomprises one or more amino acid residues selected from the groupconsisting of Lys34, Glu26, Ile38, Tyr24, His41, Glu80, Arg84, Leu106,Asp108, Glu119, Ile120, Tyr130, Lys134, Phe105, Lys137, and Arg124.

The antibody of the present invention may be a monoclonal or polyclonalantibody or portions thereof. Antigen-binding portions may be producedby recombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. In some embodiments, antigen-binding portions includeFab, Fab′, F(ab′)₂, Fd, Fv, dAb, and complementarity determining region(CDR) fragments, single-chain antibodies (scFv), chimeric antibodiessuch as humanized antibodies, diabodies, and polypeptides that containat least a portion of an antibody that is sufficient to confer specificantigen binding to the polypeptide. The antibody of the presentinvention is generated according to standard protocols. For example, apolyclonal antibody may be generated by immunizing an animal such asmouse, rat, rabbit, goat, sheep, pig, cattle, or horse with the antigenof interest optionally in combination with an adjuvant such as Freund'scomplete or incomplete adjuvant, RIBI (muramyl dipeptides), or ISCOM(immunostimulating complexes) according to standard methods well knownto the person skilled in the art. The polyclonal antiserum directedagainst the endonuclease domain of Influenza A 2009 pandemic H1N1 PA orfragments thereof is obtained from the animal by bleeding or sacrificingthe immunized animal. The serum (i) may be used as it is obtained fromthe animal, (ii) an immunoglobulin fraction may be obtained from theserum, or (iii) the antibodies specific for the endonuclease domain ofInfluenza A 2009 pandemic H1N1 PA or fragments thereof may be purifiedfrom the serum. Monoclonal antibodies may be generated by methods wellknown to the person skilled in the art. In brief, the animal issacrificed after immunization and lymph node and/or splenic B cells areimmortalized by any means known in the art. Methods of immortalizingcells include, but are not limited to, transfecting them with oncogenes,infecting them with an oncogenic virus and cultivating them underconditions that select for immortalized cells, subjecting them tocarcinogenic or mutating compounds, fusing them with an immortalizedcell, e.g., a myeloma cell, and inactivating a tumor suppressor geneImmortalized cells are screened using the H1N1 PA endonuclease domain ora fragment thereof. Cells that produce antibodies directed against theH1N1 PA endonuclease domain or a fragment thereof, e.g., hybridomas, areselected, cloned, and further screened for desirable characteristicsincluding robust growth, high antibody production, and desirableantibody characteristics. Hybridomas can be expanded (i) in vivo insyngeneic animals, (ii) in animals that lack an immune system, e.g.,nude mice, or (iii) in cell culture in vitro. Methods of selecting,cloning, and expanding hybridomas are well known to those of ordinaryskill in the art. The skilled person may refer to standard texts such as“Antibodies: A Laboratory Manual”, Harlow and Lane, Eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York (1990), which isincorporated herein by reference, for support regarding generation ofantibodies.

In another aspect, the present invention relates to the use of thecompound(s) according the present invention, the pharmaceuticalcomposition(s) according to the present invention, or the antibodyaccording to the present invention for the manufacture of a medicamentfor treating, ameliorating, or preventing disease conditions caused byviral infections with viruses of the Orthomyxoviridae family,Bunyaviridae family and/or Arenviridae family, preferably caused byviral infections with Influenza A 2009 pandemic H1N1 virus.

Said compound (i) is able to modulate, preferably to decrease, morepreferably to inhibit, the endonuclease activity of the Influenza A 2009pandemic H1N1 PA subunit or variant thereof according to the presentinvention, e.g. by binding to the endonucleolytically active site ofsaid PA subunit or variant thereof, (ii) is able to modulate, preferablyto decrease, more preferably to inhibit, the endonuclease activity ofthe Influenza A 2009 pandemic H1N1 PA subunit polypeptide fragment orvariant thereof according to the present invention, e.g. by binding tothe endonucleolytically active site of said PA subunit polypeptidefragment or variant thereof, or (iii) is able to modulate, preferably todecrease, more preferably to inhibit, the endonuclease activity of thePA subunit of other viruses, preferably of viruses of theOrthomyxoviridae family, Bunyaviridae family and/or Arenviridae family,e.g. by binding to the endonucleolytically active site of said PAsubunit.

It is preferred that said compound(s) is (are) identifiable by theabove-described methods. It is further preferred that saidpharmaceutical composition(s) is (are) producible according to theafore-mentioned methods. Thus, preferably, the present invention relatesto the use of a compound identifiable by the above-described methodsthat is able to bind to the endonucleolytically active site of theInfluenza A 2009 pandemic H1N1 PA subunit polypeptide fragment orvariant thereof, and/or is able to modulate, preferably to decrease,more preferably to inhibit the endonucleolytic activity of the InfluenzaA 2009 pandemic H1N1 PA subunit polypeptide fragment or variant thereof,or the pharmaceutical composition producible according to theafore-mentioned methods for the manufacture of a medicament fortreating, ameliorating, or preventing disease conditions caused by viralinfections with viruses of the Orthomyxoviridae family Bunyaviridaefamily and/or Arenviridae family, preferably caused by viral infectionswith Influenza A 2009 pandemic H1N1 virus.

In a further aspect, the present invention relates to the compound(s)according the present invention, the pharmaceutical composition(s)according to the present invention, or the antibody according to thepresent invention for treating, ameliorating, or preventing diseaseconditions caused by viral infections with viruses of theOrthomyxoviridae family, Bunyaviridae family and/or Arenviridae family,preferably caused by viral infections with Influenza A 2009 pandemicH1N1 virus.

Said compound (i) is able to modulate, preferably to decrease, morepreferably to inhibit, the endonuclease activity of the Influenza A 2009pandemic H1N1 PA subunit or variant thereof according to the presentinvention, e.g. by binding to the endonucleolytically active site ofsaid PA subunit or variant thereof, (ii) is able to modulate, preferablyto decrease, more preferably to inhibit, the endonuclease activity ofthe Influenza A 2009 pandemic H1N1 PA subunit polypeptide fragment orvariant thereof according to the present invention, e.g. by binding tothe endonucleolytically active site of said PA subunit polypeptidefragment or variant thereof, or (iii) is able to modulate, preferably todecrease, more preferably to inhibit, the endonuclease activity of thePA subunit of other viruses, preferably of viruses of theOrthomyxoviridae family, Bunyaviridae family and/or Arenviridae family,e.g. by binding to the endonucleolytically active site of said PAsubunit.

It is preferred that said compound(s) is (are) identifiable by theabove-described methods. It is further preferred that saidpharmaceutical composition(s) is (are) producible according to theafore-mentioned methods. Thus, preferably, the present invention relatesto a compound identifiable by the above-described methods that is ableto bind to the endonucleolytically active site of the Influenza A 2009pandemic H1N1 PA subunit polypeptide fragment or variant thereof, and/oris able to modulate, preferably to decrease, more preferably to inhibitthe endonucleolytic activity of the Influenza A 2009 pandemic H1N1 PAsubunit polypeptide fragment or variant thereof, or the pharmaceuticalcomposition producible according to the afore-mentioned methods fortreating, ameliorating, or preventing disease conditions caused by viralinfections with viruses of the Orthomyxoviridae family Bunyaviridaefamily and/or Arenviridae family, preferably caused by viral infectionswith Influenza A 2009 pandemic H1N1 virus.

In another aspect, the present invention relates to the use of thecompound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3), of the compound4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2), of the compound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1), or of the compound[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG) for the manufacture of a medicament for treating, ameliorating,or preventing disease conditions caused by viral infections withInfluenza A 2009 pandemic H1N1 virus, as the inventors of the presentinvention have determined that said compounds are able to bind to theendonucleolytically active site of the Influenza A 2009 pandemic H1N1 PAsubunit polypeptide fragment or variant thereof, and are able tomodulate, i.e. to inhibit, the endonucleolytic activity of the InfluenzaA 2009 pandemic H1N1 PA subunit polypeptide fragment or variant thereof.

In an further aspect, the present invention relates to4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3),4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2),4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1), or[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate(EGCG) for treating, ameliorating, or preventing disease conditionscaused by viral infections with Influenza A 2009 pandemic H1N1 virus.

In another aspect, the present invention relates to the use of a4-substituted 2,4-dioxobutanoic acid compound or a green tea catechinfor treating, ameliorating, or preventing disease conditions caused byviral infections with Influenza A 2009 pandemic H1N1 virus.

For treating, ameliorating, or preventing said disease conditions themedicament of the present invention can be administered to an animalpatient, preferably a mammalian patient, preferably a human patient,orally, buccally, sublingually, intranasally, via pulmonary routes suchas by inhalation, via rectal routes, or parenterally, for example,intracavernosally, intravenously, intra-arterially, intraperitoneally,intrathecally, intraventricularly, intra-urethrally intrasternally,intracranially, intramuscularly, or subcutaneously, they may beadministered by infusion or needleless injection techniques.

The pharmaceutical compositions of the present invention may beformulated in various ways well known to one of skill in the art and asdescribed above.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation administeredin the use of the present invention may be varied or adjusted from about1 mg to about 1000 mg per m², preferably about 5 mg to about 150 mg/m²according to the particular application and the potency of the activecomponent.

The compounds employed in the medical use of the invention areadministered at an initial dosage of about 0.05 mg/kg to about 20 mg/kgdaily. A daily dose range of about 0.05 mg/kg to about 2 mg/kg ispreferred, with a daily dose range of about 0.05 mg/kg to about 1 mg/kgbeing most preferred. The dosages, however, may be varied depending uponthe requirements of the patient, the severity of the condition beingtreated, and the compound being employed. Determination of the properdosage for a particular situation is within the skill of thepractitioner. Generally, treatment is initiated with smaller dosages,which are less than the optimum dose of the compound. Thereafter, thedosage is increased by small increments until the optimum effect undercircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day, if desired.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in therelevant fields are intended to be covered by the present invention.

The following figures and examples are merely illustrative of thepresent invention and should not be construed to limit the scope of theinvention as indicated by the appended claims in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 Refined atomic structure coordinates for PA polypeptidefragment amino acids 1 to 198 according to amino acids 1 to 198 of theamino acid sequence set forth in SEQ ID NO: 2 (PA H1N1 1 to 198), withor without bound compounds. For each structure there are generally fourmolecules in the crystallographic asymmetric unit (ASU) denoted A, B, Cand D. In FIGS. 1 to 5, however, only one selected molecule is shown(see below). The file header gives information about the structurerefinement. “Atom” refers to the element whose coordinates are measured.The first letter in the column defines the element. The 3-letter code ofthe respective amino acid is given and the amino acid sequence position.The first 3 values in the line “Atom” define the atomic position of theelement as measured. The fourth value corresponds to the occupancy andthe fifth (last) value is the temperature factor (B factor). Theoccupancy factor refers to the fraction of the molecules in which eachatom occupies the position specified by the coordinates. A value of “1”indicates that each atom has the same conformation, i.e., the sameposition, in all equivalent molecules of the crystal. B is a thermalfactor that measures movement of the atom around its atomic center. Thisnomenclature corresponds to the Protein Data Bank (PDB) format.

FIG. 1: (Sheets labelled FIG. 1A to FIG. 1AB) Structural co-ordinates ofthe native (i.e. without compound/ligand) H1N1 PA endonuclease domain instandard Protein Data Bank (PDB) format. Only the chain A (withassociated divalent cations, i.e. one magnesium and one manganese ion,and water molecules) from the asymmetric unit is included. Chains B, Cand D are very similar.

FIG. 2: (Sheets labelled FIG. 2A to FIG. 2AC) Structural co-ordinates ofthe H1N1 PA endonuclease domain with bound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3) in standard Protein Data Bank (PDB) format. Only thechain A (with associated divalent cations, i.e. two manganese ions, andwater molecules) from the asymmetric unit is included. The compoundEMBL-R05-3 has residue descriptor ci3.

FIG. 3: (Sheets labelled FIG. 3A to FIG. 3AB) Structural co-ordinates ofthe H1N1 PA endonuclease domain with bound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3) in standard Protein Data Bank (PDB) format. Only thechain D (with associated divalent cations, i.e. two manganese ions, andwater molecules) from the asymmetric unit is included. The compoundEMBL-05-03 has a different configuration than in chain A (FIG. 2). Saidcompound has residue descriptor ci3.

FIG. 4: (Sheets labelled FIG. 4A to FIG. 4AC) Structural co-ordinates ofthe H1N1 PA endonuclease domain with bound4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2) in standard Protein Data Bank (PDB) format. Only thechain A (with associated divalent cations, i.e. two manganese ions, andwater molecules) from the asymmetric unit is included. Chains B, C and Dare very similar. The compound EMBL-R05-2 has residue descriptor cit.

FIG. 5: (Sheets labelled FIG. 5A to FIG. 5AA) Structural co-ordinates ofthe H1N1 PA endonuclease domain with bound ribo-Uridine Monophosphate(rUMP) in standard Protein Data Bank (PDB) format. Only the chain A(with associated divalent cations, i.e. two manganese ions, and watermolecules) from the asymmetric unit is included. Chains B, C and D arevery similar. The compound rUMP has residue descriptor U.

FIG. 6: Diagram using the structural co-ordinates of FIG. 1 illustratingthe endonuclease active site (PA polypeptide fragment (chain A)),showing the divalent cations (one manganese and one magnesium) and keyactive site residues.

FIG. 7: (Sheets labelled FIG. 7A to 7B) (A) Diagram using the structuralco-ordinates of FIG. 2 illustrating the endonuclease active site (PApolypeptide fragment (chain A)), showing the bound compound EMBL-R05-3,the divalent cations (two manganese) and key active site residues thatinteract with the compound (notably Arg84) or are close to it and (B)Diagram comparing the co-ordinates of FIG. 1 (chain subjacent on lefthand side) and FIG. 2 showing change in conformation of the loop in thevicinity of Tyr24 upon binding of EMBL-R05-3 (chain A). Tyr24 side-chainmoves to partially stack with the chlorobenzene of EMBL-R05-3.

FIG. 8: Diagram using the structural co-ordinates of FIG. 3 illustratingthe endonuclease active site (PA polypeptide fragment (chain D)),showing the bound compound EMBL-R05-3, the divalent cations (twomanganese) and key active site residues that interact with the compound(notably Tyr24 and Arg84) or are close to it.

FIG. 9: Diagram using the structural co-ordinates of FIG. 4 illustratingthe endonuclease active site (PA polypeptide fragment (chain A)),showing the bound compound EMBL-R05-2, the divalent cations (twomanganese) and key active site residues that interact with the compound(notably Tyr24 and Phe105) or are close to it.

FIG. 10: Diagram using the structural co-ordinates of FIG. 5illustrating the endonuclease active site (PA polypeptide fragment(chain A)), showing the bound compound rUMP, the divalent cations (twomanganese) and key active site residues that interact with the compound(notably Tyr24 and Lys34) or are close to it.

FIGS. 11A, 11B, and 11C: 15% PAGE gels and gel filtration profile from atypical purification of H1N1 PA1-198 (SEQ ID NO:13). The arrow indicatesfaint traces of residual MBP.

FIG. 12: Frozen crystal of H1N1 PA-Nter co-crystallised with rUMP in theP212121 space-group.

FIG. 13: Divalent ion co-ordination in the native endonucleasestructure.

FIG. 14: Electron density for rUMP and divalent cations (manganese, Mn1and Mn2) in co-crystals with H1N1 PA-Nter. Refined 2Fo-Fc electrondensity contoured at 1.1 σ. Unbiased Fo-Fc electron density contoured at2.8σ. Anomalous difference map contoured at 4σ.

FIGS. 15-16: Refined atomic structure coordinates for PA polypeptidefragment amino acids 1 to 198 of the amino acid sequence set forth inSEQ ID NO: 2 with amino acids 52 to 64 replaced by the amino acidglycine (PA H1N1 1 to 198 Δ52-64: Gly (SEQ ID NO:14) with boundcompounds. For each structure there is one molecule in thecrystallographic asymmetric unit (ASU). The file header givesinformation about the structure refinement. “Atom” refers to the elementwhose coordinates are measured. The first letter in the column definesthe element. The 3-letter code of the respective amino acid is given andthe amino acid sequence position. The first 3 values in the line “Atom”define the atomic position of the element as measured. The fourth valuecorresponds to the occupancy and the fifth (last) value is thetemperature factor (B factor). The occupancy factor refers to thefraction of the molecules in which each atom occupies the positionspecified by the coordinates. A value of “1” indicates that each atomhas the same conformation, i.e., the same position, in all equivalentmolecules of the crystal. B is a thermal factor that measures movementof the atom around its atomic center. This nomenclature corresponds tothe Protein Data Bank (PDB) format.

FIG. 15: (Sheets labelled FIG. 15A to FIG. 15AB) Structural co-ordinatesof the H1N1 PA endonuclease domain with bound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1) in standard Protein Data Bank (PDB) format. The chainA (with associated divalent cations, i.e. two manganese ions, and watermolecules) from the asymmetric unit is included. The compound EMBL-R05-1has residue descriptor ci1.

FIG. 16: (Sheets labelled FIG. 16A to FIG. 16AA) Structural co-ordinatesof the H1N1 PA endonuclease domain with bound epigallocatechin 3-gallate(EGCG) in standard Protein Data Bank (PDB) format. The chain A (withassociated divalent cations, i.e. two manganese ions, and watermolecules) from the asymmetric unit is included. The compound EGCG hasresidue descriptor tte.

FIG. 17: Diagram using the structural co-ordinates of FIG. 15illustrating the endonuclease active site (PA polypeptide fragment),showing the bound compound EMBL-R05-1, the divalent cations (twomanganese) and key active site residues that interact with the compound(notably Tyr24 and Arg84) or are close to it.

FIG. 18: (Sheets labelled FIG. 18A to FIG. 18B) Diagram using thestructural co-ordinates of FIG. 16 illustrating the endonuclease activesite (PA polypeptide fragment), showing the bound compound EGCG, thedivalent cations (two manganese ions) and key active site residues thatinteract with the compound or are close to it. (B) Bound EGCG, thedivalent cations (two manganese ions) and key active site residues thatinteract with the compound or are close to it. Interactions less than3.3 Å (grey dotted lines), additional possible interactions less than 4Å (dark grey dotted lines).

FIG. 19: Superposition of all diketo inhibitor compounds (EMBL-R05-1,EMBL-R05-2, and EMBL-R05-3A and D) and EGCG bound in PA active site. Asshown in FIG. 19, the mode of binding of the three diketo inhibitors tothe metals is conserved (although there is some variability in exactposition) but the two ‘arms’ of each compound are inserted intodifferent combinations of the pockets 1 to 4. EMBL-R05-1 has a similarconfiguration to EMBL-R05-3D, with the two arms occupying pockets 2 and3. EMBL-R05-3A occupies pockets 2 and 4. EMBL-R05-2, which differsnotably from R05-1 and R05-3 in the point of substitution on thepiperidinyl ring (Table 2) occupies pocket 3 and uniquely pocket 1 (FIG.7B). The green tea compound EGCG occupies pockets 3 and 4. More potentand specific compounds could be perhaps designed that occupy more thantwo of the pockets.

EXAMPLES

The Examples are designed in order to further illustrate the presentinvention and serve a better understanding. They are not to be construedas limiting the scope of the invention in any way.

1. Methods

1.1 Cloning, Expression and Purification of PA-Nter (PA H1N1 1 to 198(SEQ ID NO:13)) and PA-Nter Mutant (PA H1N1 1 to 198 Δ52-64: Gly (SEQ IDNO:14)) from Influenza Strain a/California/04/2009-H1N1

The DNA coding for PA-Nter (residues 1-198 SEQ ID NO:13) (see SEQ ID NO:1 and 2) from influenza strain A/California/04/2009-H1N1 was synthesized(PA H1N1 1 to 198 (SEQ ID NO:13)) and sub-cloned in the expressionvector pESPRIT002 (EMBL) by GeneArt, Regensburg, Germany. The sequencewas designed to contain a MGSGMA (SEQ ID NO: 3) polypeptide linkerbetween the tobacco etch virus (TEV) cleavage site at the N-terminus toobtain 100% cleavage by TEV protease.

To further improve crystallisation properties, a deletion of part of theflexible loop (52-73) was engineered by site directed mutagenesis. Tothis end, a PCR amplification of the whole vector containing thewild-type gene was performed using two primers flanking the mutationsite, one of them phosphorylated, and TurboPfu polymerase (Stratagene).Subsequently template vector was digested with DpnI (New EnglandBiolabs) and the mutated vector was re-ligated. In the PA-Nter mutant,the amino acid sequence encompassing amino acids 52-64 was replaced by asingle glycine (PA H1N1 1 to 198 Δ52-64: Gly (SEQ ID NO:14)).

The wild-type and mutant plasmids were transformed to E. coli BL21(DE3)(Stratagene) and the protein was expressed in LB medium overnight at 20°C. after induction at an OD 0.8-1.0 with 0.2 mMisopropyl-β-thiogalactopyranoside (IPTG). The protein was purified by animmobilized metal affinity column (IMAC). A second IMAC step wasperformed after cleavage by the His-tagged TEV protease, followed by gelfiltration on a Superdex 75 column (GE Healthcare). Finally, the proteinwas concentrated to 10-15 mg/ml. See FIG. 11.

1.2 Compounds

Compounds used for co-crystallisation are given in Table 2. CompoundrUMP was purchased from Sigma. Compounds EMBL-R05-1, EMBL-R05-2, andEMBL-R05-3 (first described in {Tomassini, 1994 #397}) were customsynthesised by Shanghai ChemPartner. EGCG was purchased from Sigma(E4143).

1.3 Crystallization

Initial sitting drop screening was carried out at 20° C. mixing 100 nLof protein solution (15 mg/mL) with 100 nL of reservoir solution using aCartesian robot at the EMBL Grenoble crystallization platform. Around600 conditions were screened. Subsequently, larger crystals wereobtained at 20° C. by the hanging drop method mixing protein andreservoir solutions in a ratio of 1:1. The protein solution contained10-15 mg/mL of PA-N-ter in 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MnCl₂,2 mM MgCl₂. The refined reservoir compositions for native crystals andco-crystallization with different compounds/ligands are listed inTable 1. For co-crystallisations, compounds/ligands rUMP, EMBL-R05-3 andEGCG were added to the protein solution to final concentrations of 5 mM,1.5 mM and 5 mM, respectively. Native crystals and those co-crystallizedwith EMBL-R05-3 and EGCG were flash frozen in liquid nitrogen in theirreservoir solution with additional 25% glycerol as cryo-protectant.Co-crystals with rUMP were frozen in their reservoir solution withadditonal rUMP at 10 mM concentration and additional 20% glycerol ascryoprotectant. The structure with EMBL-R05-2 was obtained by soakingco-crystals of PA-N-ter and rUMP for 2 h with reservoir solutioncontaining 1.5 mM EMBL-R05-2 and no rUMP (the inhibitor displaces therUMP) followed by cryo protection in reservoir solution containing 20%glycerol and 1.5 mM EMBL-R05-2. FIG. 12 shows a typical co-crystal withrUMP. The structure with EMBL-R05-1 was obtained by soaking co-crystalsof PA-N-ter mutant and dTMP for 2 h with reservoir solution containingthe inhibitor followed by cryo protection in reservoir solutioncontaining 20% glycerol and the inhibitor. See also Table 2 for thecompounds/ligands.

TABLE 1 Summary of crystallisation conditions and crystallographicparameters for various compounds Compound/ Resolution Fragment/ Ligand(final Crystallisation Space group and Refinement R- FIG.(s)concentration) reservoir condition unit cell parameters factor/R-free PAH1N1 No 1.6M sodium C2  2.1 Å 1 to 198 compound formate 4 Molecules/ASU0.222/0.268 fragment (native) 0.1M HEPES pH 7 263.630 66.240 66.32 (SEQID 5% Glycerol 90.00 95.98 90.00 NO: 13) (chain A), see FIGS. 1 and 6 PAH1N1 EMBL-R05-3 2.0M ammonium P 2₁ 2₁ 2₁  2.5 Å 1 to 198 (1.5 mM)sulphate 4 Molecules/ASU 0.205/0.276 fragment 0.1M BisTris 54.57 122.54129.78 (SEQ ID pH 5.5 90.00 90.00 90.00 NO: 13) (chain A), see FIGS. 2and 7 PA H1N1 fragment (chain D), see FIGS. 3 and 8 PA H1N1 EMBL-R05-20.1M ammonium P 2₁ 2₁ 2₁ 2.07 Å 1 to 198 (1.5 mM, sulphate 4Molecules/ASU 0.205/0.259 fragment (SEQ soaked into 0.1M Bis-Tris 56.59120.81 128.20 ID NO: 13) crystals pH 5.5 90.00 90.00 90.00 (chain A),initially 25% (w/v) PEG see FIGS. 4 grown with 3350 and 9 rUMP) PA H1N1rUMP 0.1M ammonium P 2₁ 2₁ 2₁ 2.05 Å 1 to 198 (5.0 mM) sulphate 4Molecules/ASU 0.206/0.246 fragment 0.1M Bis-Tris 54.94 120.11 128.05(chain A), see pH 5.5 90.00 90.00 90.00 FIGS. 5 and 25% (w/v) PEG 103350 PA H1N1 EMBL-R05-1 25-30% PEG4K P 6₂22 (180)  1.9 Å 1-198 (1.5 mM,0.1M Tris pH 8.5 1 molecule/ASU 0.201/0.251 Δ52-64: Gly soaked into 0.2MNaCl a = b = 75.06 Fragment (SEQ crystals c = 120.05 Å ID NO: 14)initially (chain A) grown with see FIGS. 15 dTMP) and 17 PA H1N1 EGCG10% peg 3350, P6₄22 (181) 2.65 Å 1-198 (5 mM) 0.1M NaCl, 0.1M 1molecule/ASU 0.250/0.304 Δ52-64: Gly Hepes, pH 7.0 a = b = 99.9 Fragment(SEQ c = 82.7 Å ID NO: 14) (chain A) see FIGS. 16 and 18

TABLE 2 Compounds used Compound CA Index Name Formula Chemical structureEMBL-R05-3 (Tomassini et al. Antimicrob Agents Chemother 1994, 38:2827-2837) 4-[3-[(4- chlorophenyl)methyl]-1- (phenylmethyl)-3-piperidinyl]-2-hydroxy-4- oxo-2-butenoic acid C23 H24 Cl N O4

EMBL-R05-2 (Tomassini et al. Antimicrob Agents Chemother 1994, 38:2827-2837) 4-[4-[(4- chlorophenyl)methyl]-1- (cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4- oxo-2-butenoic acid C23 H30 Cl N O4

rUMP (ribo- uridine monophosphate) 5'-Uridylic acid C9 H13 N2 O9 P

EMBL-R05-1 (Tomassini et al. Antimicrob Agents Chemother 1994, 38:2827-2837) 4-[3-[(4- chlorophenyl)methyl]-1- (phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4- oxo-2-butenoic acid C23 H24 Cl N S O6

(−)- Epigallocatechin gallate (EGCG) [(2R,3R)-5,7-dihydroxy-2- (3,4,5-trihydroxyphenyl)chroman- 3-yl]3,4,5- trihydroxybenzoate C22 H18 O11

1.4 Crystal Structure Determination

Diffraction data were collected on various beamlines at the EuropeanSynchrotron Radiation Facility. Data sets were integrated with XDS andscaled with XSCALE. Subsequent data analysis was performed with theCCP4i programme suite. The initial H1N1 structure was solved withmolecular replacement with PHASER using the previously determined H3N2PA N-ter structure (Dias et al., Nature 2009). Subsequent co-crystalstructures were determined with PHASER using the H1N1 structure.Refinement was carried out with REFMAC and/or model building with COOTor O. In the C2 and P2₁2₁2₁ crystal forms there are four molecules perasymmetric unit. However due to structural variations between themolecules due to plasticity (in particular the 53-73 region) and thegenerally good resolution, NCS restraints were not applied.

2. Results

2.1 PA H1N1 Polypeptide Fragment or PA H1N1 Polypeptide Fragment VariantGeneration and Crystallisation

The inventors of the present invention found that a polypeptide fragmentcomprising amino acids 1 to 198 of SEQ ID NO: 2 of influenza strainA/California/04/2009-H1N1 (2009 pandemic strain) (PA H1N1 1 to 198)readily crystallised with and without relevant compounds/ligands (seeFIGS. 1 to 10). Crystallisation properties could further be improvedwith a polypeptide fragment variant comprising amino acids 1 to 198 ofSEQ ID NO: 2 of influenza strain A/California/04/2009-H1N1 (2009pandemic strain) with amino acids 52 to 64 replaced by the amino acidglycine (PA H1N1 1 to 198 Δ52-64: Gly) (see FIGS. 15 to 18).

Thus, in contrast to the numerous unsuccessful attempts undertaken inthe prior art, the inventors of the present invention were able toobtain structures of PA H1N1 polypeptide fragments or PA H1N1polypeptide fragment variants with and without compounds/ligands.

The structures of PA H1N1 polypeptide fragments or PA H1N1 polypeptidefragment variants with and without compounds/ligands are describedbelow. All these structures show in detail how these compounds/ligandsbind directly to the metal ions as well as interacting with a number ofresidues in the active site. Furthermore several of the interactingresidues change conformation upon ligand binding, information which wasunavailable before. This three-dimensional knowledge of the ligandinteracting residues and the regions of plasticity of the active site iscritical for the optimised design of modifications to existinginhibitors to improve their potency or for structure based design andoptimisation of novel inhibitors that effectively block endonucleaseactivity.

2.2 H1N1 PA Native Structure

Structural co-ordinates of the native (i.e. without compound/ligand)H1N1 PA endonuclease domain (PA H1N1 1 to 198(SEQ ID NO:13)) (chain A)in standard Protein Data Bank (PDB) format are shown in FIG. 1. Only thechain A (with associated divalent cations, i.e. one magnesium and onemanganese ion, and water molecules) from the asymmetric unit isincluded. Chains B, C and D are very similar. A diagram using thestructural co-ordinates of FIG. 1 illustrating the endonuclease activesite (PA polypeptide fragment (chain A)) is shown in FIG. 6. It showsthe divalent cations (one manganese and one magnesium) and key activesite residues.

2.3 EMBL-R05-3-Bound Structure

Structural co-ordinates of the H1N1 PA endonuclease domain (PA H1N1 1 to198(SEQ ID NO:13)) (chain A) with bound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3) in standard Protein Data Bank (PDB) format are shownin FIG. 2. Only the chain A (with associated divalent cations, i.e. twomanganese ions, and water molecules) from the asymmetric unit isincluded. The compound EMBL-R05-3 has residue descriptor ci3. A diagramusing the structural co-ordinates of FIG. 2 illustrating theendonuclease active site (PA polypeptide fragment (chain A)) is shown inFIG. 7A. It shows the bound compound EMBL-R05-3, the divalent cations(two manganese) and key active site residues that interact with thecompound (notably Arg84) or are close to it.

A diagram comparing the co-ordinates of FIG. 1 (chain subjacent on lefthand side) and FIG. 2 showing change in conformation of the loop in thevicinity of Tyr24 upon binding of EMBL-R05-3 (chain A) is shown in FIG.7B. The loop around Tyr24 is poorly ordered in the native structure.Tyr24 side-chain moves to partially stack with the chlorobenzene ofEMBL-R05-3. This indicates a plasticity of the active site and aninduced fit mode of ligand binding. An important conclusion fordesigning more potent inhibitors is to ensure that the extensions(‘arms’) to any ion-binding scaffold optimise interactions in one ormore pocket(s). Imperfect matching will lead to residual flexibility andsub-optimal potency, as seems to be the case for the current compounds,none of which exhibit very well defined, full occupancy binding modes.

Further, structural co-ordinates of the H1N1 PA endonuclease domain (PAH1N1 1 to 198(SEQ ID NO:13)) (chain D) with bound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-3) in standard Protein Data Bank (PDB) format are shownin FIG. 3. Only the chain D (with associated divalent cations, i.e. twomanganese ions, and water molecules) from the asymmetric unit isincluded. The compound EMBL-05-3 has a different configuration than inchain A (FIG. 2). Said compound has residue descriptor ci3. A diagramusing the structural co-ordinates of FIG. 3 illustrating theendonuclease active site (PA polypeptide fragment (chain D)) is shown inFIG. 8. It shows the bound compound EMBL-R05-3, the divalent cations(two manganese) and key active site residues that interact with thecompound (notably Tyr24 and Arg84) or are close to it.

2.4 EMBL-R05-2-Bound Structure

Structural co-ordinates of the H1N1 PA endonuclease domain (PA H1N1 1 to198 (SEQ ID NO:13)) (chain A) with bound4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-2) in standard Protein Data Bank (PDB) format are shownin FIG. 4. Only the chain A (with associated divalent cations, i.e. twomanganese ions, and water molecules) from the asymmetric unit isincluded. Chains B, C and D are very similar. The compound EMBL-R05-2has residue descriptor cit. A diagram using the structural co-ordinatesof FIG. 4 illustrating the endonuclease active site (PA polypeptidefragment (chain A)) is shown in FIG. 9. It shows the bound compoundEMBL-R05-2, the divalent cations (two manganese) and key active siteresidues that interact with the compound (notably Tyr24 and Phe105) orare close to it.

2.5 Ribo-Uridine Monosphosphate (rUMP)-Bound Structure

Structural co-ordinates of the H1N1 PA endonuclease domain (PA H1N1 1 to198 (SEQ ID NO:13)) (chain A) with bound ribo-Uridine Monophosphate(rUMP) in standard Protein Data Bank (PDB) format are shown in FIG. 5.Only the chain A (with associated divalent cations, i.e. two manganeseions, and water molecules) from the asymmetric unit is included. ChainsB, C and D are very similar. The compound rUMP has residue descriptor U.

Co-crystallisation trials with rUMP gave large, well-ordered crystals ina new orthorhombic space-group (FIGS. 12 and 14).

A diagram using the structural co-ordinates of FIG. 5 illustrating theendonuclease active site (PA polypeptide fragment (chain A)) is shown inFIG. 10. It shows the bound compound rUMP, the divalent cations (twomanganese) and key active site residues that interact with the compound(notably Tyr24 and Lys34) or are close to it. The rUMP binds with twooxygens of the phosphate completing the co-ordination sphere of Mn1, oneof them also co-ordinating Mn2. The base is well stacked on Tyr24 andLys34 makes a hydrogen bond to the 02 position. The ribose hydroxylgroups do not make hydrogen bonds to the protein, consistent with thefact that deoxy ribose binds equally well and the protein is a DNAase asmuch as an RNAase {Dias, 2009 #448}.

The conformation observed for rUMP is quite different from thatpreviously published (PDB entry 3hw3 {Zhao, 2009 #444}). The latterstructure was obtained by soaking nucleotides into existing crystals ofthe endonuclease in the absence of manganese and the electron density isvery poor. In this structure, a water molecule replaces Mn1 and amagnesium ion replaces Mn2. This difference in metal ligation isreflected in the altered conformation of Glu119. The ribose and basepositions are quite different from the positions in the structureprovided herein and unable to interact with Lys34 or Tyr24. Thedifferences between the two structures may reflect firstly the lack ofmanganese and secondly the fact that soaking pre-grown crystals does notallow the active site to adapt to the ligand as is more likely the casefor co-crystallisation.

2.6 EMBL-R05-1-Bound Structure

Structural co-ordinates of the H1N1 PA endonuclease domain (PA H1N1 1 to198 Δ52-64: Gly (SEQ ID NO:14)) with bound4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoicacid (EMBL-R05-1) in standard Protein Data Bank (PDB) format are shownin FIG. 15. The chain (with associated divalent cations, i.e. twomanganese ions, and water molecules) from the asymmetric unit isincluded. The compound EMBL-R05-1 has residue descriptor ci1. A diagramusing the structural co-ordinates of FIG. 15 illustrating theendonuclease active site (PA polypeptide fragment) is shown in FIG. 17.It shows the bound compound EMBL-R05-1, the divalent cations (twomanganese) and key active site residues that interact with the compound(notably Tyr24 and Arg84) or are close to it.

2.7 EGCG-Bound Structure

Epigallocatechin 3-gallate (EGCG), is the ester of epigallocatechin andgallic acid and is the most abundant catechin in green tea. It hasrecently been reported that EGCG inhibits the influenza endonuclease{Kuzuhara, 2009 #629}. Co-crystallisation of PA H1N1 1 to 198 Δ52-64:Gly (SEQ ID NO:14) with (−)-Epigallocatechin gallate gave a new crystalform (Table 1) diffracting to 2.65 Å resolution. The compound wasclearly observed in the active site. Strong extra density also existsaround a 2-fold crystallographic axis and most likely represents otherEGCG molecules trapped by crystal packing.

Structural co-ordinates of the H1N1 PA endonuclease domain (PA H1N1 1 to198 Δ52-64: Gly (SEQ ID NO:14)) with bound epigallocatechin 3-gallate(EGCG) in standard Protein Data Bank (PDB) format is shown in FIG. 16.The chain (with associated divalent cations, i.e. two manganese ions,and water molecules) from the asymmetric unit is included. The compoundEGCG has residue descriptor tte. A diagram using the structuralco-ordinates of FIG. 16 illustrating the endonuclease active site (PApolypeptide fragment) is shown in FIG. 18A. It shows the bound compoundEGCG, the divalent cations (two manganese ions) and key active siteresidues that interact with the compound or are close to it. FIG. 18Bshows the bound EGCG, the divalent cations (two manganese ions) and keyactive site residues that interact with the compound or are close to it.Interactions less than 3.3 Å (grew dotted lines), additional possibleinteractions less than 4 Å (dark grey dotted lines) are shown.

2.8 Superposition of all Diketo Inhibitor Compounds

A superposition of all diketo inhibitor compounds (EMBL-R05-1,EMBL-R05-2, and EMBL-R05-3A and D) and EGCG bound in PA active site isshown in FIG. 19. As shown in FIG. 19, the mode of binding of the threediketo inhibitors to the metals is conserved (although there is somevariability in exact position) but the two ‘arms’ of each compound areinserted into different combinations of the pockets 1 to 4. EMBL-R05-1has a similar configuration to EMBL-R05-3D, with the two arms occupyingpockets 2 and 3. EMBL-R05-3A occupies pockets 2 and 4. EMBL-R05-2, whichdiffers notably from R05-1 and R05-3 in the point of substitution on thepiperidinyl ring (Table 2) occupies pocket 3 and uniquely pocket 1 (FIG.7B). The green tea compound EGCG occupies pockets 3 and 4. More potentand specific compounds could be perhaps designed that occupy more thantwo of the pockets.

2.9 Divalent Cation Binding

In the native structure (FIG. 1 and FIG. 6), two divalent cations areobserved in the active site. These are identified (using magnitude ofelectron density and anomalous scattering, data not shown) to be amanganese atom in site 1 (Mn1 in FIG. 13), ligated by His41, Asp108,Glu119, Ile120 and two water molecules (W4 and W5) and a magnesium ionin site 2 (Mg2 in FIG. 13), ligated by Glu80 and Asp108 and four watermolecules (W1, W2, W3 and W4). In the other structures (FIGS. 2-5 andFIGS. 7-10 as well as FIGS. 15-18) the ions in both site 1 and site 2are manganese ions (Mn1 and Mn2), as identified by magnitude of electrondensity and anomalous scattering (e.g. for rUMP, see FIG. 14).

The invention claimed is:
 1. A polypeptide fragment comprising an N-terminal fragment of the PA subunit of a viral RNA-dependent RNA polymerase possessing endonuclease activity, wherein said PA subunit has at least 95% sequence identity to SEQ ID NO: 2 having a maximum length of 240 amino acids, wherein the N-terminus is identical to or corresponds to amino acid position 1 and the C-terminus is identical to or corresponds to an amino acid at a position selected from positions 190 to 198 of the amino acid sequence of the PA subunit according to SEQ ID NO: 2, or is a variant thereof, wherein said variant comprises the amino acid serine at an amino acid position 186 according to SEQ ID NO: 2 or at an amino acid position corresponding thereto.
 2. The polypeptide fragment of claim 1, which is soluble and remains in the supernatant after centrifugation for 30 min at 100,000×g in an aqueous buffer under physiologically isotonic conditions.
 3. The polypeptide fragment of claim 1, which is crystallizable.
 4. The polypeptide fragment of claim 1, wherein the N-terminal fragment has at least amino acids 1 to 190 of the PA subunit of the RNA-dependent RNA polymerase of Influenza A 2009 pandemic H1N1 virus according to SEQ ID NO:
 2. 5. The polypeptide fragment of claim 1, wherein said polypeptide fragment is purified to an extent to be suitable for crystallization and is at least 85% pure.
 6. The polypeptide fragment of claim 1 to which two divalent cations are bound.
 7. The polypeptide fragment of claim 6, wherein the divalent cation is manganese and/or magnesium.
 8. The polypeptide fragment of claim 1, which (a) consists of amino acids 1 to 198 of the amino acid sequence set forth in SEQ ID NO: 2 and of an N-terminal linker having the amino acid sequence MGSGMA (SEQ ID NO: 3) and which has the structure defined by (i) the structure coordinates as shown in FIG. 1, (ii) the structure coordinates as shown in FIG. 2, (iii) the structure coordinates as shown in FIG. 3, (iv) the structure coordinates as shown in FIG. 4, or (v) the structure coordinates as shown in FIG. 5, or (b) consists of amino acids 1 to 198 of the amino acid sequence set forth in SEQ ID NO: 2 with amino acids 52 to 64 replaced by the amino acid glycine and of an N-terminal linker having the amino acid sequence MGSGMA (SEQ ID NO: 3) and which has the structure defined by (vi) the structure coordinates as shown in FIG. 15, or (vii) the structure coordinates as shown in FIG.
 16. 9. The polypeptide fragment of claim 8, wherein the polypeptide fragment having the structure defined by (i) has a crystalline form with space group C2 and unit cell dimensions of a=26.36 nm±0.5 nm, b=6.62 nm±0.3 nm, c=6.63 nm±0.3 nm, α=90 deg, β=96±2 deg, γ=90 deg, (ii) to (v) has a crystalline form with space group P2₁2₁2₁ and unit cell dimensions of a=5.46±0.3 nm, b=12.25±0.4 nm, c=13.0±0.3 nm, α=90 deg, β=90 deg, γ=90 deg, (vi) has a crystalline form with space group P6₂22 and unit cell dimensions of a=7.50 nm±0.3 nm, b=7.50 nm±0.3 nm, c=12.00 nm±0.5 nm, α=90 deg, β=90 deg, γ=120 deg, or (vii) has a crystalline form with space group P6₄22 and unit cell dimensions of a=9.99 nm±0.5 nm, b=9.99 nm±0.5 nm, c=8.27 nm±0.3 nm, α=90 deg, β=90 deg, γ=120 deg.
 10. The polypeptide fragment of claim 8, wherein the crystal diffracts X-rays to a resolution of 2.6 Å, 2.1 Å, 1.9 Å or higher.
 11. The polypeptide fragment of claim 8, wherein the polypeptide fragment has a crystalline form, and the crystal diffracts X-rays to a resolution of 2.1 Å or higher.
 12. The polypeptide fragment of claim 8, wherein the polypeptide fragment has a crystalline form, and the crystal diffracts X-rays to a resolution of 1.9 Å or higher.
 13. A polypeptide consisting of the amino acid sequence according to SEQ ID NO:14.
 14. The polypeptide of claim 13, wherein the polypeptide is in a crystal form selected from the group consisting of a crystal in space group P6₂22 with unit cell dimensions of a=75.0 Å±3 Å, b=75.0 Å±3 Å, c=120.0 Å±5 Å, α=90 deg, β=90 deg, γ=120 deg, or a crystal in space group P6₄22 with unit cell dimensions of a=99.9 Å±5 Å, b=99.9 Å±5 Å, c=82.7 Å±3 Å, α=90 deg, β=90 deg, γ=120 deg. 